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A  Bacteriologic  Study  of  the  Diphtheroid  Organisms  with 
Special  Reference  to  Hodgkin's  Disease 


DISSERTATION 

Submitted  in  Partial  Fulfilment  of  the  Requirements  for  the  Degree 

of  Doctor  of  Philosophy  in  the  Faculty  of  Pure  Science 

of  Columbia  University  in  the  City  of  New  York 


BY 

FREDERICK   EBERSON,  B.Sc,  M.A.,  M.Sc. 


NEW  YORK  CITY 
1918 


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PREFACE 

The  study  reported  in  the  following  pages  was  undertaken  with  the 
purpose  of  sifting  out  the  voluminous  data  which  have  accumulated 
on  the  subject  of  diphtheroid  organisms. 

The  greatest  impetus  given  to  such  an  investigation  has  resulted,  in 
recent  years,  from  the  attempts  of  numerous  workers  to  associate 
Hodgkin's  disease  with  a  certain  organism  belonging  to  the  diphtheroid 
group.  In  a  short  time  the  literature  on  the  subject  assumed  great 
proportions. 

A  survey  of  the  field  revealed  immediately  the  fact  that  the  group 
as  a  whole  has  been  indiscriminately  used  as  a  cover  for  any  organ- 
ism which  bore  some  sort  of  morphological  resemblance,  real  or 
apparent,  to  the  diphtheria  type.  The  material  presented  in  this  study 
is  by  no  means  exhaustive  since  a  great  many  methods  will  have  to  be 
tried  before  a  clear-cut  answer  is  to  be  given  to  the  question  as  tc 
what  a  true  diphtheroid  is  and  where  it  belongs  in  the  comprehensive 
group  of  which  these  bacteria  are  members. 

Of  great  moment  is  the  idea  of  mutation  introduced  by  Rosenow 
and  his  school.  This  problem  is  fraught  with  such  difficulties  not 
amenable  to  satisfactory  control,  that  a  reply  to  the  findings  of  these 
workers  will  have  to  be  deferred.  At  present  the  outlook  for  a  deci- 
sive answer  to  this  question  seems  to  be  found  in  at  least  one  method 
whereby  we  can  control  the  constitution  of  our  nutrient  media  by 
means  of  H-ion  concentration,  and  also  in  a  phase  of  serological 
technic  which  has  not  been  studied  heretofore.  In  the  following 
pages  an  effort  will  be  made  to  attack  the  problem  from  these  angles. 

I  wish  to  thank  Professor  Hans  Zinsser  for  much  valuable  advice 
and  suggestions  and  Dr.  R.  E.  Buchanan  of  the  Iowa  State  College 
for  his  criticisms  on  nomenclature.  F.  E. 

Bacteriological    Laboratory.   Columbia    University. 
New  York  City,  January,  1918. 


93754 


CONTENTS 


Preface     3 

1.  NOMENCLATURE     AND     CLASSIFICATION     OF     THE     DIPH- 

THEROIDS 

Introduction     7 

HISTORICAL   REVIEW    OF    DIPHTHEROIDS    AND    EMENDATIONS 8 

DISTRIBUTION    OF    DIPHTHEROIDS 18 

Diphtheroids    in    Hodgkin's    Disease 17,  18 

Diphtheroids    in    Glands    and    Tissues 18 

Cultural  Study  and  Grouping  of  Organisms 18,  19 

Diphtheroids   from   the   Eye 21 

Nasal    Diphtheroids    21 

Diphtheroids  from  Ascitic  Fluids 21 

Diphtheroids  in  Blood  Cultures 22 

Skin    Diphtheroids    22 

Diphtheroids  from  the  Brain 23 

Diphtheroids   from  the  Appendix 23 

Source  and   Fermentative    Reactions 24 

CLASSIFICATION     OF     THE     DIPHTHEROIDS 25 

2.  SEROLOGIC   STUDY 

EXPERIMENT     WITH     HODGKIN's     STRAINS 30 

Agglutination    Tests    31 

Complement    Fixation    31 

APPLICATION     OF     AGGLUTININ     ABSORPTION     TESTS     TO     DIFFERENTIATION 

OF    TYPE    DIPHTHEROIDS 32 

RELATION     OF     TYPE     DIPHTHEROIDS 33,   34 

3.  PLEOMORPHISM  OF  DIPHTHEROIDS,  MUTABILITY  OF  TYPES 

AND  A  METHOD  FOR  DETERMINING  MUTANTS 

VARIABILITY    OF    BACTERIA 34 

CULTURAL     STUDY     OF     MUTA'TION .' 36 

Experiments  with  Museum  Strain  of  C.  enzymicus  (Mellon)....  36 

Effect  of  Various  Mediums  on  Selection  of  Type Zl 

Experiments  with  Mellon  Strain  cultivated  on  Blood  Medium..  39 

STUDY    WITH    PURE   LINE    OF    C.    ENZYMICUS 39 

Bacillary    Type    39 

"Transition"  of  Bacillus  to   Coccus 39 

Coccus    Type     40 

Behavior  of  Coccus  and  Bacillary  Types  in  Carbohydrates 41 

SEROLOGIC     EXPERIMENTS      42 

Absorption    of    Agglutinins 42 

Absorption  Experiments  with  Coccus  Serum 42 

Absorption  Experiments  with  Bacillary  Serum 43 

SUMMARY    AND    CONCLUSIONS 44 

Bibliography  45,  46,  47 

Plates  1-3.     Explanation  of  plates 48,  49,  51,  53 

Vita    55 


A     BACTERIOLOGIC     STUDY     OF     THE     DIPHTHEROID 

ORGANISMS     WITH     SPECIAL    REFERENCE    TO 

HODGKIN'S     DISEASE 

Plates    1-3 

Frederick     Eberson 

From  the  Department  of  Bacteriology,  Columbia  University,  College  of  Physicians  and  Sur- 
geons, New   York   City 

I.    NOMENCLATURE    AND    CLASSIFICATION    OF    THE 
DIPHTHEROIDS 

Introduction 

Within  the  past  few  years  the  literature  dealing  with  organisms  of 
the  diphtheria  group  have  received  many  new  contributions.  It  seems 
that  most  human  and  animal  diseases  of  hitherto  unknown  etiology 
have  been  ascribed  to  some  sort  of  diphtheroid  organism.  Whether  or 
not  these  results  will  be  confirmed  has  little  bearing  on  the  problem 
which  confronts  the  systematic  bacteriologist  who  finds  himself  hard 
put  to  it  in  attempting  to  classify  the  great  number  of  organisms  which 
for  no  reason  other  than  a  slight  resemblance  to  the  diphtheria  bacteria 
have  been  thrown  haphazardly  into  a  group  of  "unknowns" — the 
diphtheroids. 

Since  Loeftler's  -discovery  of  the  diphtheria  organism,  so  many 
so-called  "pseudodiphtheria"  bacteria  have  been  mentioned  in  the  litera- 
ture that  it  seems  necessary  at  this  time  to  survey  the  field  and  come  to 
an  understanding  as  to  what  constitutes  a  diphtheroid. 

In  a  recent  paper  on  the  "Nomenclature  of  the  Coccaceae," 
Buchanan  has  pointed  out  the  chaotic  state  of  bacterial  nomenclature. 
That  the  naming  of  species,  genera  and  higher  groups  needs  careful 
study  and  revision  is  a  foregone  conclusion.  A  great  need  is  felt  for 
such  an  analysis  in  the  groups  of  bacteria  which  possess  common  char- 
acteristics.    Such  a  group  is  the  diphtheroid  group. 

First,  it  is  necessary  to  define  a  "diphtheroid."  The  term  "pseudo- 
diphtheria"  has  always  seemed  unsatisfactory  because  the  organisms 
so-called  dififer  in  so  many  respects  from  the  true  Klebs-Loeffler  organ- 
ism as  to  warrant  the  dropping  of  the  term  from  the  literature  except 
in  conditions  to  be  suggested  herewith.     A  review  of  the  extensive 

Received  for  publication  Feb.  6,   1918. 


8  Frederick  Eberson 

work  done  in  this  group  reveals  the  fact  that  there  are  bacteria  which 
resemble  the  true  diphtheria  organism  morphologically  and  culturally, 
but  which  differ  in  one  important  respect,  namely,  that  of  virulence  as 
tested  on  guinea-pigs.  The  true  Klebs-Loeffler  has  been  shown  to  fer- 
ment dextrose,  maltose  and  dextrin  with  acid  production  and  to  possess 
definite  toxic  properties  on  injection,  resulting  in  the  death  of  the 
animal  in  3-4  days  with  a  characteristic  inflammation  and  congestion 
of  the  suprarenal  glands.  To  those  bacteria  which  lack  this  virulence 
but  which  otherwise  conform  to  the  character  of  true  diphtheria  bac- 
teria, should  be  assigned  the  species  designation  ''pseudodiphtheria." 
The  generic  name  given  to  the  group  by  Lehmann  and  ^eumann  is 
Corynebacterium  and  should  be  retained  for  the  organisms  of  this 
group.  Certain  characters  ought  to  be  accepted  as  properly  belonging 
to  the  species  before  we  can  adequately  define  what  we  mean  by  a  diph- 
theroid. These  may  be  enumerated  as  follows :  Morphological  resem- 
blance to  diphtheria  organism,  gram-positive,  nonmotile,  absence  of 
spores,  presence  or  absence  of  metachromatic  granules,  no  gas-produc- 
tion in  carbohydrate  mediums.  The  fermentation  of  sugars  may  or 
may  not  occur.  This  property  gives  us  a  basis  for  classification  which 
will  be  taken  up  later.  A  careful  study  of  the  different  species  which 
have  been  described  shows  that  the  characters  enumerated  ought  rightly 
to  be  accepted  as  a  basis  for  differentiating  the  diphtheroids  from  other 
bacteria  which  have  been  improperly  classed  with  them.  With  this  end 
in  view  the  different  species  are  taken  up  individually  and  examined 
from  the  standpoint  of  validity  and  priority.  The  suitability  of  the 
name  is  considered  from  the  viewpoint  of  sufficient  description,  and  in 
the  light  of  more  comprehensive  studies  which  have  given  us  sugges- 
tions for  proper  nomenclature.  When  the  species  name  implies  etiology 
which  is  unknown,  doubtful  or  unconfirmed,  that  part  of  it  is  modified 
to  suit  the  case. 

Historical  Review  of  Diphtheroids  and  Emendations 
B.  pseudodiphtheriticum  (Loeffler)  Migula 
The  description  is  given  as  follows :  Gram-positive,  no  polar  bodies,  non- 
motile.  Grows  luxuriantly  on  blood  serum  with  a  glistening  layer.  In  broth 
clouding  is  produced.  LoeiBer  described  a  pseudodiphtheria  organism  in  his 
first  paper  on  the  etiology  of  diphtheria.  He  said  the  organism  resembled 
diphtheria  but  was  avirulent  for  guinea-pigs ;  that  it  was  smaller  than  the  true 
diphtheria  and  showed  a  marked  absence  of  club-shaped  forms.  In  1888, 
V.  Hofmann  published  an  account  of  avirulent  diphtheria-like  organisms  iso- 
lated from  the  oral  cavity  in  26  of  45  normal  persons.  He  called  these  organ- 
isms pseudodiphtheria  bacilli  and  described  them  as  follows :  Clouding  pro- 
duced in  broth,  no  acid  production,  grow  at  room  temperature,  no  spores. 


Diphtheroid  Organisms  9 

The  rightful  name  for  this  species  is  Corynebacterium  pseudodiphtheriticum 
(Hofmann-Wellenhof).  It  is  questionable,  however,  if  we  can  retain  this  name 
for  all  diphtheroids  which  are  morphologically  and  culturally  like  the  one 
described  by  v.  Hofmann.  Recent  studies  show  that  there  are  diflferent  species 
of  saprophytic  diphtheroids  which  conform  to  the  type  characters,  yet  are  not 
found  in  the  nose  or  throat.  At  the  close  of  this  communication  we  shall 
describe  a  few  such  strains  which  have  been  isolated  from  widely  varying 
sources. 

B.    PSEUDODIPHTHERITICUS    ALCALIFACIENS 
B.    PSEUDODIPHTHERITICUS    ACIDUM    FACIENS 

These  names  were  applied  by  'Kurth  to  diphtheroid  organisms  which  he  found 
to  be  avirulent,  and  were  capable  of  producing,  respectively,  a  weakly  alkaline 
and  an  acid  reaction  in  sugar  broth.  The  alcalifaciens  species  conforms  to  the 
following  description :  No  granules,  short  forms  on  serum  with  white  growth, 
abundant  growth  in  bouillon  with  clouding,  no  acid  produced  in  sugar  broth. 

This  description,  although  meager,  suggests  that  Kurth  was  undoubtedly 
dealing  with  the  type  Hofmanni.  B.  alcalifaciens  should  lapse  into  synonymy 
with  this  species.  As  for  the  B.  pseud,  acidum  faciens  a  new  species  is  hardly 
justified  in  view  of  the  fact  that  avirulent  diphtheroids  differ  in  their  fermenta- 
tive properties  in  various  sugars.  Moreover  the  description  is  incomplete. 
Apart  from  the  interest  which  attaches  to  the  first  attempt  at  classifrcation 
from  the  fermentation  standpoint,  these  two  species  have  no  particular  value. 

B.    XEROSIS     (neISSER    AND    KUSCHBERT)     MIGULA 

Club-shaped,  plump,  and  longer  septate  forms.  On  blood  serum  terminal 
swellings  never  noted.  Gram-positive,  polar  bodies  sometimes  visible,  no 
growth  below  25  C,  no  clouding  in  bouillon,  but  granular  sediment  is  formed. 
Distinguished  from  pseudodiphtheria  by  its  ability  to  grow  only  above  25  C. 

One  point  needs  slight  modification  in  this  description  of  B.  xerosis,  namely, 
that  of  temperature  requirement.  On  certain  mediums,  such  as  ascitic  dextrose 
agar  we  have  noted  growth  of  12  strains  of  diphtheroids  which  were  isolated 
fiom  the  eye.  These  organisms  complied  with  the  description  given  for 
B.  xerosis  as  regards  morphology  and  fermentative  properties  in  different  car- 
bohydrate broths.  Some  of  the  strains  developed  very  slowly  at  temperatures 
ranging  from  18-25  C,  showing  delicate  growth  on  slant  mediums  after  4 
days.     Eisenberg  states  that  B.  xerosis  grows  only  at  body  temperature. 

B.  septatum  gelpke 

Nonmotile,  gram-positive,  club-shaped  forms  on  serum.  Young  cultures 
show  polar  staining,  no  spores.  Grows  on  serum  with  a  delicate  yellowish 
dry  appearance.  In  bouillon  no  clouding  occurs  but  a  granular  flaky  deposit 
is  visible.  Does  not  grow  at  temperatures  below  28  C.  Is  to  be  distinguished 
from  other  organisms  of  the  group  by  this  property.  Associated  with  conjunc- 
tival inflammation — "schwelltjngskatarrh." 

A  comparison  of  the  2  organisms  just  described  makes  it  appear  questionable 
that  we  are  dealing  with  a  distinct  species  in  B.  septatum.  This  organism 
which  appears  to  be  identical  with  B.  xerosis  is,  like  the  latter,  not  necessarily 
associated  with  a  conjunctivitis  for  B.  xerosis  has  been  isolated  frequently 
from  normal  eyes.  Zinsser  is  of  the  opinion  that  the  B.  xerosis  is  a  harmless 
parasite  which  may  occur  more  often  in  the  slightly  inflamed  than  in  the  nor- 
mal conjunctiva.     The  term  septatum  is  not  especially  characteristic  for  diph- 


10  Frederick  Eberson 

theroids  associated  with  normal  and  diseased  eyes.  Large  striped  or  septate 
bacteria  have  been  found  in  the  nose  and  in  the  blood  during  the  course  of  per- 
sonal study  on  this  group  of  organisms. 

B.     NODOSUS     PARVUS     (lUSTGARTEN-MANNABERG) 

Club-shaped  organism,  disposed  at  angles  or  parallel  nonmotile,  spores 
absent,  slow  growth  at  Zl  C.  on  agar  with  white  appearance,  nonpathogenic, 
facultative.  Found  in  normal  human  urethra.  Migula  has  changed  the  name 
of  this  bacterium  somewhat  and  calls  it  B.  nodosum.  Similar  organisms  have 
been  isolated  from  the  urine.  In  one  case  we  have  found  this  organism  asso- 
ciated with  a  gonorrheal  urethritis  and  in  another  instance  in  normal  urine. 
Marked  differences  have  been  noted  in  the  acid-forming  properties  of  these  2 
strains.  One  gives  rise  to  abundant  acid  in  several  sugars  and  the  other  fails 
to  attack  any  of  them.  They  are  gram-positive.  Hine  mentions  11  strains  of 
diphtheroids  which  he  isolated  from  the  urogenital  tract.  They  appeared  as 
large  clubbed  forms,  markedly  segmented  and  exhibited  granules,  often  of  large 
size.    They  fermented  dextrose,  saccharose,  maltose  and  dextrin. 

It  is  suggested  that  the  name  Corynebact.  nodosum  (Migula)  be  given  to  these 
organisms  and  that  B.  nodosum  parvum  lapse  into  synonymy. 

B.    ENDOCARDITIS    GRISEUS     (  WEICHSELBAUM) 

Flugge  classifies  this  species  under  the  head  of  pseudodiphtheria.  It  is  a 
short  rod,  morphologically  resembling  B.  xerosis.  Grows  well  at  room  temper- 
ature, facultative,  gram-positive,  irregular  staining  property,  js  actively  motile, 
glistening  growth  on  mediums,  with  grayish-white  appearance.  In  streak  cul- 
tures a  light  brown  or.  reddish-gray  growth  is  observed.  Pathogenic  for 
guinea-pigs  and  white  mice.  Rabbits,  when  injected,  show  endocarditis.  Found 
on  the  heart  valves  in  a  fatal  case  of  recurrent  ulcerative  endocarditis.  Accord- 
ing to  the  suggested  definition  for  diphtheroid  this  bacillus  should  be  ruled  out. 
The  property  of  motility  has  not  been  observed  among  members  of  the  diph- 
theria group. 

B.  erythematis 

Demme,  in  1887,  described  an  organism  which  caused  erythema  with  death 
in  guinea-pigs.  It  is  a  delicate  rod,  disposed  in  smaller  or  larger  groups,  non- 
motile,  forms  spores,  grows  best  at  Vl  C.  but  is  slow  in  development,  facultative, 
gram-positive.  Flugge  has  put  this  bacterium  in  the  diphtheroid  group.  Apart 
from  the  diphtheroid  picture  which  might  be  seen  in  a  microscopic  field  because 
of  the  configuration  in  groups  and  by  virtue  of  the  bulging  forms  due  to  spores, 
there  is  no  apparent  reason  for  classifying  this  species  as  a  diphtheroid.  Spore- 
formation  rules  the  organism  out  of  the  group. 

B.    RENALIS    BOVIS    (eNDERLEN)     MIGULA 

Rods  with  thickened  ends,  nonmotile,  gram-positive.  In  broth,  a  granular 
precipitate  is  formed,  the  mediums  remaining  clear.  Obligate  aerobe.  Does 
not  grow  at  room  temperature.  A  more  adequate  description  is  given  by  Ernst. 
He  isolated  the  strain  from  cases  of  pyelonephritis  in  cattle.  The  organism 
grows  poorly  as  compared  with  the  diphtheria  bacterium.  Babes-Ernst  gran- 
ules appear  much  later.  It  is  club-shaped,  lancetlike  or  cylindrical.  No  growth 
takes  place  at  room  temperature.  Nq  acid  produced  in  dextrose  and  glycerin, 
no  spores,  facultative  aerobe,  nonmotile,  gram-positive.  Pathogenicity  appears 
doubtful.     Ernst  was  unable  to  demonstrate  virulence  for  guinea-pigs  or  rab- 


Diphtheroid  Organisms  11 

bits  by  means  of  intraperitoneal  or  intrapulmonary  inoculations.  Concludes 
that  the  organism  is  not  etiologic  for  pyelonephritis,  since  he  could  not  recover 
the  organism  from  the  lesions  or  reproduce  the  disease. 

Grips  named  an  organism  which  he  isolated  from  swine  Bacillus  pyogenes 
'  suis.  Kiinnemann  described  a  similar  organism  from  cattle  under  the  name 
B.  pyogenes  hovis.  Glage  combined  the  two  under  the  single  specific  name 
B.  pyogenes.  Glage  and  Prieve,  in  their  studies  have  shown  that  the  organisms 
are  related  to  the  influenza  bacillus  and  are  members  of  the  group  of  hemo- 
globinophilic  organisms.  The  name  C.  pj'Ogenes  (Glage)  is  perhaps  most  suit- 
able for  this  organism. 

Among  human  beings,  pus  cavities  and  suppurating  wounds  have  been  found 
to  harbor  diphtheroid  organisms.  Whether  or  not  these  possess  pyogenic  prop- 
erties is  speculative.  It  may  be  of  interest  to  note  that  three  strains  isolated 
by  the  writer  have  been  obtained  from  an  anal  pus  pocket  in  a  patient  whose 
kidney  underwent  suppuration  and  complete  necrosis.  These  organisms  have 
been  injected  subcutaneously  and  intraperitoneally  into  guinea-pigs  with  nega- 
tive results.  Diphtheroids  found  in  sinuses  following  abscess  formation  are 
not  rare.    It  is  very  likely  that  these  organisms  are  skin  inhabitants. 

B.    STRIATUS    FLAVUS     (V.    BESSER) 

Eisenberg's  description :  Found  in  normal  nasal  mucus.  Short  thick  rods, 
sometimes  curved,  with  striped  or  barred  appearance.  On  agar  white,  thick 
growth  with  yellow  pigment.     Grows  rapidly  at  room  temperature,  no  spores. 

B.    STRIATUS    ALBUS     (v.    BESSER) 

Same  source  as  B.  striatus  flavus.  Appearance  the  same.  Growth  on  agar 
is  white  and  glistening,  no  spores.  Fliigge  mentions  B.  striatus  albus  only. 
Chester  has  named  the  organism  B.  striatum. 

That  these  striped  diphtheroids  occur  in  the  normal  nasal  mucus  has  been 
shown  by  several  investigators.  Undoubtedly  v.  Besser  was  dealing  with  but 
one  type  of  organism  which  is  capable  of  producing  pigment  varying  from  white 
to  yellow.  In  a  comprehensive  study  made  by  Morse  this  was  found  to  be  the 
case.  Our  own  studies  have  confirmed  these  findings.  Morse  has  given  this 
species  diphtheroid  the  name  B.  flavidus  with  the  following  diagnosis :  Thick 
forms  with  clear  cut  bars.  Large  and  irregular  granules  visible.  Heavy 
growth  on  serum  with  pigment  varying  in  color  from  white  to  yellow.  Ferments 
dextrose  and  maltose  but  not  saccharose.  B.  flavidus  as  a  species  designation 
is  invalid  since  a  prior  description  had  already  been  given  by  v.  Besser  and 
later  amended  by  Chester  to  conform  with  binomial  system  of  nomenclature. 
The  emendation  by  Morse  is  recognized  but  the  species  should  be  written, 
Coryn.  striatum   (Chester)  nov.  comb. 

B.    BORDONI — UFFREDUZZI     (eISENBERG) 

This  is  known  as  B.  epidermidis  and  has  been  described  as  Bizzozeros  Lep- 
tothrix  epidermidis.  It  is  an  avirulent  spore-former  growing  best  at  15-20  C 
Since  this  organism  does  not  conform  to  the  genus  requirements,  it  should  not 
be  classed  with  the  diphtheroids. 

B.    CLAVATUS 

Fliigge,  in  1894,  gave  this  name  to  a  club-shaped  bacillus  which  he  isolated 
from  milk  heated  to  100  C.  for  one  hour.  It  is  a  long  bacillus,  motile,  forms 
spores.    Swollen  at  ends,  anaerobic,  liquefies  gelatin. 


12  Frederick  Eberson 

This  same  species  is  met  with  in  an  earlier  work  on  dysentery  by  Kruse 
and  Pasquale.  They  isolated  from  the  heart  blood  and  mesenteric  fluid  in 
oases  of  Egyptian  dysentery  an  organism  which  they  call  "B.  clavatus."  It  is 
not  described  in  any  way  as  to  warrant  its  recognition  as  a  species.  The  char- 
acters enumerated  by  Fliigge  make  it  invalid  as  a  type  of  corynebacteria. 

SPORE-FORMING    DIPHTHEROID   OF    DE    SIMONI 

Isolated  from  nasal  secretion  in  case  of  ozena.  Said  to  form  spores  only 
when  grown  in  milk  or  on  potato.  Facultative  anaerobe.  Grows  luxuriantly 
in  dextrose,  lactose  and  maltose  broth  with  slight  acid  formation. 

The  author  was  undoubtedly  misled  by  the  appearance  of  an  impure  culture. 
The  illustration  given  in  his  article  shows  a  complete  transformation  from  a 
typical  diphtheroid  of  slender  and  unevelnly  staining  aspect  to  a  blunt  spore- 
bearing  rod  in  older  cultures.  This  is  highly  improbable,  yet  were  it  absolutely 
authentic,  one  should  not  be  inclined  to  class  this  species  with  the  diphtheria 
group  for  reasons  outlined  previously. 

B.    DIPHTHEROIDES 

Described  by  Klein.  Isolated  from  purulent  secretion  of  udder  of  cow. 
Organism  fails  to  grow  below  25  C.  Growth  on  agar  slow  and  sparse,  aerobic. 
On  serum  grows  best  at  Zl  C.,  producing  white  colonies.  It  is  oval  or  spherical 
in  form  with  a  deeply  staining  center.  Club-shaped  forms  present,  gram- 
positive.  Produces  abscess  in  guinea-pigs  after  subcutaneous  or  intraperito- 
neal inoculation.  The  organism  dies  within  a  week  when  grown  on  artificial 
mediums. 

This  diphtheroid,  according  to  the  few  cultural  characters  given,  is  in  all 
likelihood  identical  with  C.  pyogenes  (Glage).  The  description  is  too  meager 
to  warrant  retaining  it  as  a  distinct  species.  The  fact  that  the  organism  died 
so  readily  on  artificial  mediums  indicates  that  it  resembled  the  hemoglobino- 
philic  type  of  which  Glage's  is  an  example. 

B.   VARIABILIS   LYMPHAE   VACCINALIS 

This  is  the  first  of  several  descriptions  of  a  species  associated  with  the  vac- 
cine pustules  of  calves.  Nakanishi,  in  1900,  reported  on  an  organism  which  he 
isolated  from  a  vaccine  pustule.  It  is  club-shaped,  coccoid,  segmented  or 
branched.  Stains  poorly  with  Gram,  facultative  anaerobe,  nonmotile.  Central 
granule  noted  when  stained  with  warm  fuschin.  Growth  on  serum  is  abundant. 
Pale  yellow  or  orange-yellow  pigment  produced.  Poor  growth  on  gelatin. 
Spore-formation  is  questionable.  The  name  of  this  species  is  not  a  binomial 
and  therefore  may  be  discarded.  A  somewhat  later  and  more  adequate  descrip- 
tion by  Levy  and  Fickler  is  invalid,  however,  for  the  same  reason.  Their 
organisms  were  found  to  be  identical  with  that  of  Nakanishi  and  possessed 
pyogenic  properties  for  guinea-pigs,  mice  and  rabbits.  The  name  given  to  the 
species  by  these  authors  is  Corynebact.  lymphae  vaccinalis.  It  is  definitely 
gram-positive.  In  1904  this  species  is  met  with  as  Coryneb.  pyogenes,  so 
described  by  Lewandowsky  who  published  a  paper  almost  simultaneously  with 
Galli-Valerio.  The  last  named  author  found  his  species  to  be  identical  with 
Nakanishi's  and  that  of  Levy  and  Fickler.  Galli-Valerio  proposes  the  name 
Coryneb.  vaccinae  (Galli-Valerio).  Lewandowsky's  designation  is  a  binomial 
and  valid.  Galli-Valerio  gave  no  reasons  for  rejecting  a  prior  description  and 
was  apparently  unaware  of  Lewandowsky's  work  which  appeared  at  the  same 


Diphtheroid  Organisms  13 

time.  The  species  is  best  described  perhaps  as  Coryneb.  vaccinae  (Galli- 
\'alerio)  because  the  name  C.  pyogenes  had  already  been  used  by  Glage  (q.  v.) 
for  an  organism  isolated  from  cattle. 

B.  coryzae  segmentosus  Cautley ;  B.  diphtheroides  citreus ;  B.  diphtheroides 
brevis;   B.  maculatus ;   B.   diphtheroides   liquefaciens ;   B.   auris ;    B.  ceruminis. 

These  seven  strains  were  described  by  Graham  Smith.  He  isolated  the  first 
5  from  the  nose  and  mouth,  and  the  last  2  from  the  ear.  B.  coryzae  segmentosus 
was  first  cited  by  Cautley  in  a  local  English  government  report.  The  organism 
is  characterized  as  follows :  Nonmotile,  gram-positive,  polar  bodies  visible, 
white  growth  in  gelatin.  In  broth,  clear  with  sediment.  Slight  acid  produced 
in  dextrose  bouillon.    Avirulent. 

The  species'  name  is  objectionable  from  the  etiologic  standpoint  and  violates 
the  rule  for  binomial  nomenclature.  This  organism  has  been  isolated  from 
normal  nasal  mucus  on  numerous  occasions  and  is  not  necessarily  associated 
with  coryza.  It  differs  from  C.  striatum  in  its  slight  acid-producing  power  in 
dextrose,  its  slow  growth  and  small  polar  bodies.  That  this  species  may  be 
obtained  from  the  eye  as  well  as  from  the  nose  has  been  noted  by  the  writer. 

In  these  instances  it  is  to  be  differentiated  from  C.  xerosis  by  the  absence 
of  acidity  when  grown  in  maltose  and  saccharose  broths.  As  a  rule  C.  xerosis 
ferments  these  last  named  sugars  (Morse).  It  is  suggested  that  the  name 
Coryneb.  segmentosum  nom.  nov.  be  substituted  for  the  invalid  B.  coryzae  seg- 
mentosus Cautley.  B.  diphtheroides  citreus  corresponds  to  Coryn.  striatum 
(Chester).  It  produces  yellow  pigment.  The  name  given  by  Graham  Smith 
is  not  a  binomial  and  disregards  priority.  It  should  lapse  into  synonymy  with 
Coryn.  striatum   (Chester)  Eberson. 

B.   DIPHTHEROIDES   BREVIS 

From  pus  of  oral  abscess,  Growth  on  agar,  white  and  slimy.  In  gelatin, 
white  and  dry,  clouding  in  bouillon,  granular  sediment.  Highly  acid  in  sugar 
broth.  This  species  will  be  discussed  later  in  connection  with  diphtheroids 
isolated  from  the  ear.  This  organism  B.  maculatus  is  meagerly  described  as  an 
acid-producer.  Avirulent.  It  is  impossible  to  tell  wherein  this  organism 
agrees  with  or  differs  from  the  main  types  which  have  been  established  in 
recent  studies  and  for  that  reason  can  hardly  be  looked  on  as  a  distinct  species 
until  more  complete  biometrical  study  is  made  for  strains  from  a  similar 
source. 

B.   DIPHTHEROIDES    LIQUEFACIENS 

On  serum  growth  is  light  yellow.  Very  long  bacillus,  somewhat  curved, 
with  polar  bodies.  Slightly  motile;  gram-positive;  on  agar,  growth  is  thick 
and  moist  with  light  yellow  pigment.  Gelatin  is  liquefied  in  3  days.  In  bouillon, 
slight  clouding  with  granular  sediment.  In  sugar  broth  reaction  is  weakly 
alkaline  or  neutral.  Avirulent.  A  search  through  the  literature  indicates  that 
gelatin  liquefying  properties  have  not  been  associated  with  diphtheroids,  except 
in  this  one  instance.  In  61  strains  studied  by  the  writer,  but  one  was  found 
to  liquefy  gelatin.  This  species  will  be  described  under  isolations  from  ascitic 
fluids  and  Graham  Smith's  organism  will  be  discussed  and  emended. 

B.   AURIS 

From  aural  pus  of  scarlet  fever  patient.  On  serum  is  similar  to  that  of 
Klebs-Loeflfler  organism,  but  slower  in  growth.  Segmented,  nonmotile,  gratn- 
positive,  polar  bodies  visible.    On  agar,  gray  colonies ;  on  gelatin,  small  trans- 


14  Frederick  Eberson 

lucent  colonies  which  become  white  in  three  days.     Bouillon  is  clouded  slightly 
and  sediment  produced.    Visible  acid  produced  in  sugar  broth. 

B.    CERUMINIS 

From  normal  ear.  Long,  thin,  curved  organism.  Gram-positive,  polar 
bodies.  Bouillon  remains  clear  with  sediment.  Neutral  or  alkaline  reaction 
in  sugar  broth.    Avirulent. 

DIPHTHEROIDS    FROM    THE    EAR 

Diphtheroids  of  the  ear  have  been  made  the  subject  of  a  very  valuable  paper 
by  Alice  Hamilton.  In  a  study  of  over  50  cases  of  otitis  media  and  scarlet 
fever  she  succeeded  in  differentiating  2  groups  of  organisms. 

Group  1. — Includes  diphtheroids  with  the  following  characters  :  Short  rods, 
gram-positive  (weakly  staining).  Abundant  growth  on  agar.  Ferment  dex- 
trose and  saccharose  but  not  maltose  or  dextrin.     Nonpathogenic. 

Group  2. — Long,  barred  or  granular,  often  clubbed.  Gram-positive,  scanty 
growth  on  agar.  Ferment  dextrose  and  maltose,  but  not  saccharose.  Often 
virulent  for  guinea-pigs. 

The  pathogenicity  of  these  organisms  is  altogether  different  from  that  of  the 
diphtheria  bacterium  inasmuch  as  diphtheria  antitoxin  did  not  protect  against 
them.  Hamilton's  studies  showed  also  that  the  organisms  isolated  from  otitis 
undoubtedly  have  some  bearing  on  the  disease  since  vaccine  treatment  had  a 
marked  effect  on  opsonic  determinations.  If  we  compare  this  type  (Group  2) 
with  the  B.  auris  described  by  Graham  Smith  it  appears  that  we  have  the 
same  organism.  Unfortunately  the  work  of  the  last  named  author  does  not 
mention  virulence  tests  and  the  strains  were  too  few  in  number  to  merit  com- 
parison. We  are  safe,  however,  in  recognizing  that  the  diphtheroids  found  in 
the  ear  are  not  of  the  same  type  and  that  in  cases  of  otitis  media  and  post- 
scarlatinal infections  another  species,  not  necessarily  of  etiologic  significance, 
seems  to  predominate.     The  name  Coryn.  auris   (Graham  Smith)   is  proposed. 

B.    CERUMINIS 

This  organism  has  been  observed  also  by  Dr.  Dwyer,  of  this  laboratory. 
This  organism  fails  to  ferment  any  of  the  sugars  and  differs  from  C.  Hofmanni 
in  morphology  as  well  as  its  behavior  in  broth  which  remains  clear,  with  sedi- 
ment.    The  name  should  be  Coryn.  ceruminis    (Graham  Smith)   comb.  nov. 

There  remains  for  consideration  the  diphtheroids  of  Hamilton's  Group  I. 
These  organisms  differ  from  C.  hoagii  (Morse)  only  in  the  absence  of  a  salmon- 
pink  pigment.  In  all  likelihood  these  strains  conform  to  this  type.  Besides 
these  ear  diphtheroids,  there  seems  to  be  a  variety  which  is  characterized  by 
vigorous  fermentative  properties  in  a  number  of  sugars.  Two  such  strains  have 
been  isolated  by  the  writer.  A  description  follows :  Gram-positive,  small  rods, 
abundant,  white  glistening  growth  on  agar,  no  spores.  Form  large  amounts 
of  acid  in  dextrose,  lactose,  saccharose,  maltose,  mannite,  and  dextrin,  but  do 
not  split  raffinose.  Acidity  titrations  with  N/20  NaCH  after  48  hours'  incuba- 
tion were  as  follows : 


Dextrose 

Lactose 

Saccharose  Maltose 

Mannite 

Dextrin 

Strain    1 

3.2 

3.5 

3.9               4.0 

2.8 

2.3 

Strain    2 

5.0 

3.6 

4.4               4.7 

2.6 

2.0 

These  strains  were  obtained  from  cases  of  otitis  media.    This  species  is  similar 
to  the  diphtheroides  brevis  described  by  Graham  Smith.     It  is  suggested  that  a 


Diphtheroid  Organisms  15 

binomial  be   used   for  the   species  with  the   emendation   noted   above,   namely, 
Coryn.  acidum  sp.  nov. 

B.    PSEUDODIPHTHERITICUS    GAZOGENES 

This  is  the  only  gas-forming  diphtheroid  met  with  in  the  literature.  Jacobson 
has  described  it  as  follows :  Isolated  from  stools  of  an  infant.  Is  a  vigorous 
gas  former,  club-shaped  with  thickened  ends.  "One  of  the  cultures  resembled 
streptobacilli.  This  character  did  not  persist  in  the  transplants."  Gram-positive 
and  "in  cultures  a  few  days  old  gram-negative  organisms  were  observed."  No 
spores.  Good  growth  in  gelatin,  after  5-6  days.  Litmus  milk  is  reddened  in 
24  hours.  Nonpathogenic.  From  the  description  given  by  the  author  it  does 
not  seem  likely  that  we  have  to  deal  with  one  organism.  Several  things  point 
to  the  impurity  of  the  culture.  Isolation  from  feces,  as  was  here  the  case,  makes 
such  contamination  all  the  more  probable.  According  to  the  definition  of  a 
diphtheroid,  this  organism  which  is  here  described  is  hardly  to  be  reckoned  as 
one  of  the  genus  Corynebacterium. 

DIPHTHEROIDS  AND  ACNE  VULGARIS 

Unna,  in  1891,  repeatedly  found  in  lesions  of  acne  vulgaris  an  organism  which 
he  concluded  was  the  cause  of  the  disease.  He  did  not  isolate  the  organism  in 
pure  culture,  but  gave  it  the  name  "Flaschenbacillus"  and  described  it  as  fol- 
lows :  Unevenly  staining  rod  having  clubbed  ends  and  showing  ellipsoid  forms. 
In  1894,  Hodara,  in  a  bacteriologic  study  of  acne,  reported  the  presence  of  two 
types  of  bacteria  in  acne  lesions.  One  type  is  that  of  Unna  and  the  other 
Hodara  considers  as  another  variety  which  is  similar  in  appearance  but  shows 
varying  morphology.  For  this  species  he  used  the  name  "Flaschen-Kugel- 
Bacillus"  ("Bacillus  a  Flacons-Ballons"). 

Neither  of  these  papers  goes  into  any  detail  concerning  the  organisms  and 
are  of  little,  if  any,  value  for  classification  purposes.  The  first  accurate  report 
on  these  diphtheroids  is  given  by  Sabouraud  about  3  years  later.  Sabouraud 
studied  the  bacterial  flora  of  the  sebaceous  plugs  which  can  be  squeezed  out  of 
the  skin.  He  found  an  organism  which  he  claimed  to  be  the  true  cause  of 
the  disease  according  to  his  definition,  and  which  he  described  as  follows : 
Punctiform  bacillus,  almost  coccoid,  gram-positive,  less  than  1  mikron  in  length. 
Stains  readily  with  anilin  dyes.  Requires  highly  acid  mediums  for  growth. 
Produces  intense  clouding  in  broth.  Sabouraud  named  the  organism  "bacille 
de  seborrhee  grasse." 

It  was  not  until  1901,  when  Gilchrist  made  a  thorough  study  on  the  bacterial 
flora  of  the  skin,  that  an  adequate  description  is  found  for  the  bacteria  which 
were  mentioned  by  Unna  and  his  followers.  Gilchrist's  description  points  to 
the  identity  of  his  organism  with  that  of  his  predecessors.  It  is  gram-positive, 
stains  unevenly  and  very  small.  It  is  anaerobic,  although  many  strains  were 
found  to  grow  aerobically  after  continued  cultivation  on  artificial  culture 
mediums.  Surface  smears  made  with  the  material  from  pustules  failed  to  show 
growth  whereas  stab  cultures  and  heavily  inoculated  unbroken  pustules  always 
showed  growth.  The  organism  is  named  B.  acnes  by  the  author.  We  are  still 
in  doubt  as  to  the  cause  of  acne  and  for  this  reason  B.  acnes  as  a  species 
designation  may  be  criticized.  Fleming  claims  to  have  obtained  beneficial  results 
by  the  use  of  autogenous  vaccines  prepared  from  similar  organisms  which  he 
isolated  from  cases  of  acne.  As  Sabouraud  pointed  out  these  diphtheroids  are 
found  in  sebaceous  plugs  and  recent  observations  of  numerous  workers  as  well 
as  of  our  own,  shovv  that  these  bacteria,  anaerobic  in  character,  can  be  isolated 


16  Frederick  Eberson 

very  readily  from  "blackheads"  without  the  use  of  special  mediums.  It  is 
believed  by  some  that  "blackheads"  are  to  be  looked  on  as  a  stage  of  acne.  In 
order  to  adhere  to  the  rules  of  priority  we  should  designate  this  diphtheroid 
as  follows ;  Coryn.  acnes  (Gilchrist)  comb.  nov.  Cultural  studies  with  this 
organism  were  made  by  Siidmerson  and  Thompson  in  1910.  They  confirmed 
Sabouraud's  findings  as  to  the  suitability  of  highly  acid  mediums  for  culti- 
vating C.  acnes.  One  part  horse  serum  to  3  parts  of  3%  nutrient  agar  (4.0)  is 
recommended  as  a  culture  medium  for  isolation.  Ordinary  glucose  agar  may 
be  used  just  as  well,  however.  Fermentation  studies  show  that  the  organism 
ferments  dextrose,  saccharose  (shght),  maltose,  mannite  and  inulin.  Raffinose 
is  fermented  by  1  strain  and  not  by  another.  The  authors  suggest  raflfinose  as 
a  means  for  differentiating  2  species.  Several  strains  studied  by  the  writer 
formed  no  acid  in  raffinose  and  in  dextrin. 

Virulence  tests  were  made  on  guinea-pigs  and  mice.  Only  the  latter  suc- 
cumbed to  an  injection  of  4  loopfuls  of  a  5  day  culture.  On  necropsy  the  ani- 
mals showed  a  greatly  enlarged  spleen,  enlarged  and  congested  mesenteric  glands, 
enlarged,  soft  and  anemic  kidney,  congested  suprarenals  and  an  enlargement 
and  congestion  of  the  axillary  and  inguinal  glands. 

B.    PSEUDOTUBERCULOSIS    OVIS    PREISZ    (nOCARd) 

This  organism  was  originally  isolated  by  Preisz  and  Guinard  from  necrotic 
areas  in  the  kidney  of  sheep.  Later,  Preisz  described  the  organism  more  fully. 
It  is  gram-positive,  nonmotile  and  club-shaped.  On  serum  it  produces  a  heavy 
moist  growth  with  golden-yellow  pigment.  Toxin  production  is  attributed  to 
the  organism  and  is  fatal  for  guinea-pigs,  rabbits,  goats  and  sheep.  In  guinea- 
pigs,  intraperitoneal  inoculation  gives  rise  to  an  orchitis.  In  larger  animals 
there  is  produced  a  pseudotuberculosis  which  can  be  differentiated  from  true 
tuberculosis  by  histologic  study  of  the  pseudotubercles  which  do  not  show  the 
characteristic  giant  cells.  In  a  recent  paper  by  Hall  and  Stone  this  organism 
was  found  to  produce  suppurative  processes  throughout  the  lymphatics  in  guinea- 
pigs.  The  toxin  which  is  elaborated  by  the  bacterium  resembles  but  is  not  iden- 
tical with  that  of  diphtheria,  yet  is  partially  neutralized  by  diphtheria  antitoxin. 
The  authors  found  that  the  organism  was  hemolytic  in  blood  agar  which  did 
not  contain  an  excess  of  fermentable  carbohydrate.  Dextrose  and  maltose  were 
fermented,  but  glycerol  was  not  attacked,  thus  differing  from  C.  striatum  which 
ferments  glycerin  and  like  the  B.  pseudotuberculosis  ovis  Preisz  produces  a 
yellow  pigment.  Three  strains  of  this  organism  (horse,  sheep,  calf)  which  were 
sent  to  the  Museum  of  Natural  History  by  the  writer  were  tested  for  acid  pro- 
duction along  with  the  other  diphtheroids.  The  strain  from  the  calf  produced 
more  acid  in  dextrose  and  dextrin  than  either  of  the  other  two.  The  detailed 
results  of  the  tests  were  as  follows : 


Dextrose 

/-                 IN/, 

Lactose 

6u  aciu           ^ 
Saccharose 

Dextrin 

(Horse)           1.8 
(Sheep)           0.9 
(Calf)              2.4 

0.6 

0.3 
0.4 

0.3 
0.3 

0.7 
1.7 

Coryn  pseudotuberculosis   (Prei 

sz)   comb. 

nov.  is  perhaps 

the  correct  desig- 

nation. 

B.    HOAGII 

Morse  described  this  type  in  1912  as  representing  one  of  the  groups  which 
she  established  as  the  result  of  a  biometrical  study  of  295  strains.  The  char- 
acters may  be  summarized  as  follows :     Medium-sized  bacterium  showing  solid 


Diphtheroid  Organisms  17 

barred  and  wedged  forms  with  abundant  but  small  and  imperfect  granules.  On 
serum  it  produces  a  very  heav}',  confluent,  glistening  growth  with  a  character- 
istic salmon-pink  color.  Ferments  dextrose  and  saccharose,  but  not  maltose. 
The  organism  was  described  by  Hoag,  but  not  named  by  him.  The  species  should 
be  written  Corynebacterium  hoagi   (Morse)   comb.  nov. 

CORYNEBACTERIUM     HODGKINIl 

This  name  is  applied  to  an  organism  said  to  be  the  cause  of  a  glandular 
disease  about  which  pathologists  and  bacteriologists  are  still  at  odds.  In  recent 
years  Bunting  and  Yates,  Rosenow,  and  Billings  have  been  strong  adherents  to 
the  belief  that  Hodgkin's  disease  is  caused  by  a  diphtheroid  organism  which 
they  have  cultivated  from  glandular  material  obtained  from  such  patients. 
Organisms  similar  to  those  of  Bunting  and  Yates  have  been  isolated  from 
Hodgkin's  cases  by  Rhea  and  Falconer  recently,  and  according  to  the  earlier 
work  of  Fraenkel  and  Much,  and  de  Negri  and  Mieremet  have  been  found  in 
similar  cases.  Fraenkel  and  Much  thought  that  the  organism  was  related  to  the 
tubercle  bacillus  since  it  was  found  by  them  to  be  resistant  to  antiformin.  This 
character  has  been  shown  to  be  insignificant  by  de  Negri  and  Mieremet  who 
observed  the  contrary.  This  test  was  made  on  60  strains  of  diphtheroids  by 
the  writer  and  in  no  case  was  an  antiformin-resistant  type  observed.  The 
first  description  of  this  diphtheroid  is  given  by  Fraenkel  and  Much  as  follows: 
Granular,  antiformin-resistant,  gram-positive,  nonacid  fast.  Avirulent  for  guinea- 
pigs,  de  Negri  and  Mieremet  add  several  cultural  characters :  Plump  short  rod, 
granular,  polar  staining.  Grows  best  at  Z2  C.  and  prefers. aerobic  conditions 
and  alkaline  reaction  of  mediums.  Abundant  growth  on  Loeffler's  serum,  with 
slimy  consistency.  Bordet  medium  of  blood-glycerin,  potato-extract  and  agar 
gives  abundant  growth  in  24  hours.  According  to  Bunting  and  Yates,  gelatin 
is  not  liquefied  and  broth  is  not  clouded,  but  sediment  is  formed.  In  this  paper 
it  is  not  proposed  to  discuss  the  etiology  of  Hodgkin's  disease,  but  in  review- 
ing the  work  of  others  and  from  personal  studies,  it  seems  that  we  are  not 
yet  ready  to  ascribe  to  these  diphtheroids  the  great  importance  which  has  been 
given  to  them.  That  organisms  similar  to  these  can  be  isolated  from  normal 
individuals  as  well  as  from  a  variety  of  diseased  conditions  has  been  well  shown 
by  Bloomfield  who  made  a  study  of  the  bacterial  flora  of  lymphatic  glands.  He 
found  avirulent  organisms  which  were  correlated  with  saprophytes  on  the  body 
surface  in  cases  of  Hodgkin's  disease,  lymphosarcoma,  carcinoma.  Another 
group  of  organisms  seemed  to  possess  a  relation  to  oxygen  supply.  These 
were  of  frequent  occurrence.  They  are  short,  pleomorphic  rods,  gram-positive, 
nonacid  fast,  no  polar  bodies  and  exhibit  clubbed  forms.  Bloomfield  found 
these  in  cases  of  lymphosarcoma,  arthritis,  carcinoma  and  Hodgkin's  disease. 
Virulence  tests  on  rabbits,  guinea-pigs  and  mice  were  negative.  He  concludes 
that  definitely  diseased  glands  yield  a  greater  number  of  successful  cultures  than 
do  normal  glands.  Saprophytic  organisms  are  filtered  out  by  the  glands  and 
become  a  permanent  flora  of  the  same.  None  of  the  isolated  strains  appears 
to  be  the  etiologic  factor  in  specific  diseases.  Another  study  by  Fox  has  thrown 
further  light  on  the  question  of  secondary  invasion.  Fox  found  that  no  one 
bacterial  variety  with  definite  morphologic  and  cultural  characters  has  been 
isolated  from  cases  of  Hodgkin's  disease.  That  these  diphtheroids  may  he  found 
in  enlarged  glands  in  other  conditions  is  mentioned  by  Fox  and  has  been 
observed  by  the  writer  who  has  isolated  organisms  morphologically  and  cul- 
turally identical  with  the  so-called  C.  Hodgkinii,  from  an  hypertrophied  tonsil 
and  from  lymph  nodes  and  other  sources.     For  the  sake  of  comparison  the  fol- 


18 


Frederick  Eberson 


lowing  table  is  given  so  as  to  show  the  lack  of  uniformity  in  Hodgkin's  strains 
which  our  study  has  so  far  illustrated. 

A  glance  at  Table  1  shows  that  the  strains  isolated  from  Hodgkin's  cases 
not  only  differ  among  themselves,  but  are  not  to  be  distinguished  from  organ- 
isms obtained  from  widely  varying  sources.  The  study  of  Hodgkin's  disease 
is  to  be  the  subject  of  another  report.  Until  the  etiology  of  Hodigkin's  disease 
is  more  definitely  ascertained,  it  is  suggested  that  C.  Hodgkinii  should  not  be 
retained  as  a  valid  species  inasmuch  as  the  two  C.  Hodgkinii  (Rosenow)  strains 
are  widely  different  in  cultural  characters.  In  view  of  the  fact  that  other 
organisms  which  are  in  no  way  related  to  this  disease  or  any  adenopathy 
resembling  it,  are  culturally  and  morphologically  identical  with  this  organism, 
the  name  Hodgkinii  serves  no  purpose  and  leads  to  confusion. 

TABLE  1 
Showing    the    Lack    of    Uniformity  in    Acid    Production    in    Hodgkin's    Stkains 


N/20  Acid  Produced  in 

Cul- 
ture                   Source 

Saccha- 
Dextrose    Lactose        rose      i  Maltose 

i                                   i 

Mannite      Dextrin 

1  Hodgkin's  (Bunting)....... 

2  Hodgkin's  (Bunting) 

3  Lympiiocy tic  leukemia 

4  Acute  pseudoleukemia 

5  Hodgkin's  (Bunting) 

6  Blood  (Bunting) 

0.2 

0.7                — 
0.3                 — 
0.2                2.5 
0.2                 — 
0.2                 — 

1    1    1    1    1    1 

0.3 
1.1 
0.5 
0.5 
0.3 
0.3 
2.7 
0.2 
0.4 
0.3 
0.3 
0.2 
1.7 
2.9 
2.2 
0.3 
0.4 
1.7 

- 

0.2 
0.3 

S       C.  Hodgkinii  (Rosenow).... 
10      Blood  (Eberson) 

2.8                 - 

1.9 

0.3 

11      Tonsil  (Eberson) 

0.2                 — 

1.7 

1.7 



12  Anal  pus  pocket  (Eberson). 

13  C.  Hodgkinii  (Rosenow).... 
15       Bone  marrow  (Eberson).... 

28       Hodgkin's  (Rosenow) 

34       Hodgkin's  (Rosenow) 

38       Hodgkin's  (Rosenow) 

44      Lymph  node  (Eberson) 

49       Hodgkin's  (Eberson) 

51    1  Hodgkin's  (Eberson) 

0.1 
0.2 
0.9 
2.0 
1.9 

0.1 
1.5 

0.1 

0.3 
0.3 

0.3 

DISTRIBUTION     OF    DIPHTHEROIDS 


At  this  point  it  seems  convenient  to  tabulate  the  diphtheroids  which 
have  been  found  in  glands  and  tissues  and  arrange  the  several  species 
which  have  not  been  classified  heretofore. 


Diphtheroids   in    Glands    and   Tissues 

The  following  signs  indicate  acid  production :  ±,  0.1  to  0.5  N/20  NaOH 
required  for  neutralization;  +,  0.6  to  1.0  N/20  NaOH  required  for  neutral- 
ization ;  ++,  1.1  to  2.5  N/20  NaOH  required  for  neutralization ;  +  +  +,  2.6 
to  4.Q  N/20  NaOH  required  for  neutralization ;  — ,  no  acid ;  alk,  weakly  alkaline. 

Sugar  broths  were  prepared  as  follows:  1%  by  weight  of  each  of  the  carbo- 
hydrates were  added  to  meat  infusion  broth  prepared  according  to  standard 
methods.  The  carbohydrates  used  were  Merck's  C.  P.  lactose,  saccharose, 
maltose,  mannite  and  raffinose  and  Kahlbaum's  dextrose  and  dextrin.  Difco 
peptone  was  used  in  preparation  of  broth.  To  the  mediums  (sterilized  in 
Arnold    for  20   minutes   on   3   successive   days   and   immediately   cooled)    was 


Diphtheroid  Organisms 


19 


added  sterile  ascitic  fluid  in  the  proportions  of  1  in  10.  The  mediums  were 
then  incubated  at  37  C.  for  24  hours  and  1  day  at  room  temperature  to  test 
for  sterility.  For  inoculation,  48-hour  cultures  of  the  diphtheroids  grown  on 
ascitic  dextrose  agar  (0.5%  dextrose)  were  used.  Titrations  were  made  in  the 
cold  after  48  hours'  incubation,  using  N/20  NaOH.  Five  c  c  of  the  broth  were 
used  for  titration.  Instead  of  adding  45  c  c  of  water  and  a  few  drops  of  phenol- 
phthalein  to  each  5  c  c  of  material,  it  was  found  more  convenient  to  add  phenol- 
phthalein  to  a  large  volume  of  water  and  then  to  make  this  exactly  neutral 
by  addition  of  a  small  amount  of  N/1  NaOH,  as  was  necessary  for  the  water 
used  in  the  tests.  In  this  way,  errors  due  to  variations  in  reaction  of  dis 
tilled  water  may  be  avoided.  This  method  and  the  procedure  followed  in  the 
preparation  of  mediums  were  adhered  to  in  all  titrations. 


TABLE   2 
The  Acid-Producing  Properties  of  Diphtheroids  Found  in  Glands  and  Tissues 


Cul- 
ture 


Source 


Dex- 
trose 


Lac- 
tose 


B.*  1  j  Hodgkin's  disease ± 

B.   2    Hodgkin's  disease -t- 

B.   3    Axillary  gland,  lymphocytic  leu- 
kemia   ± 

B.   i    Cutaneous  tumor,  acute  pseudo- 
leukemia   ± 

B.    5    Hodgkin's  disease ± 

B.   6    Hodgkin's  disease ± 

B.   8    C.  Hodgkinii +  +  + 

8t  Hypertrophied  tonsil — 

10  Blood ± 

11  I  Hypertrophied  tonsil ± 

B.  13     C.  Hodgkinii ± 

13t  Hypertrophied  tonsil — 

14tl  Tonsil — 

15    Bone  marrow,  leukemia ± 

27t  Hypertrophied  tonsil — 

B.  28    Hodgkin's  disease + 

29    Hypertrophied  tonsil — 

R.  34    Hodgkin's  disease +-1- 

35    Hypertrophied  tonsil ± 

B.  38    Hodgkin's  disease +  + 

44     Lymph  node — 

47  :  Brain,  meningitis -f-l--l- 

49    Hodgkin's  disease ± 

51     Hodgkin's  disease I  ++ 


+  +  + 


Baffl- 
nose 


-f 

± 

+  +  + 


alk 


alk. 


+-t- 


+  -f- 

+  +  + 

+  + 

+  + 
± 
+  + 


—  alk. 


alk. 
alk. 


*  B.  =:  Bunting  strains;  B.  =  Bosenow  strains. 

Diphtheroids  isolated  from  glands  and  certain  tis.sues  fall  into  sev- 
eral groups  :  ( 1 )  Those  which  ferment  dextrose  and  maltose  slightly ; 
(2)  those  which  ferment  dextrose,  saccharose,  maltose  vigorously  and 
dextrin  slightly;  (3)  those  which  ferment  dextrose  and  maltose  vigor- 
ously; (4)  those  which  ferment  dextrose  and  maltose  vigorously  and 
dextrin  slightly;  (5)  those  which  ferment  lactose,  maltose  and  rafifinose 
vigorously,  and  dextrin  and  dextrose  slightly;  (6)  those  which  ferment 
lactose  and  maltose  slightly;  (7)  those  which  ferment  none  of  the 
sugars ;  and  (8)  those  which  ferment  dextrose,  lactose  and  maltose 
vigorously.     That  the  range  of  fermentative  powers  varies  widely  is 


20  Frederick  Eberson 

seen  at  a  glance.  Before  any  systematic  classification  is  attempted 
many  more  strains  must  be  isolated  from  lymph  glands  and  tissues.  A 
few  new  strains  will  be  described  here,  however. 

The  diphtheroids  which  were  isolated  from  tonsils,  of  which  224  were 
studied,  varied  in  morphology  from  very  small  rods  to  large  club-shaped  forms. 
They  were  all  gram-positive,  nonmotile,  nonacid  fast,  nonspore  forming,  aerobic, 
and  failed  to  produce  gas  in  carbohydrate  mediums.  Four  were  hemoglobino- 
philic.  A  few  of  those  strains  possessed  hemolytic  properties.  One  produced 
an  orange  pigment  when  grown  on  ascitic  dextrose  agar  slants.  This  property 
was  observed  on  ordinary  agar  as  well.  Diphtheroids  with  orange  pigment 
had  not  been  described  in  the  literature  at  this  time,  and  it  is  suggested  that 
this  organism  be  called  C.  aurantiacum.  A  similar  species  was  obtained  from 
a  Hodgkin's  gland.  The  pigment  production  was  studied  from  the  standpoint 
of  oxygen  and  temperature  requirements.  At  room  temperature  (18-25  C.) 
under  aerobic  and  anaerobic  conditions,  pigment  was  produced  abundantly  in 
from  24-48  hours.  At  Zl  C.  under  aerobic  conditions,  the  orange  color  was 
abundant  in  the  same  interval  of  time,  whereas  under  anaerobic  conditions, 
a  very  slight  coloration  was  observed  after  10-14  days.  The  organism  is 
gram-positive,  nonmotile,  does  not  form  spores,  is  not  acid  fast  and  is  faculta- 
tive. On  ascitic  dextrose  agar,  it  grows  abundantly  with  an  orange  tinge  after 
24  hours.  It  is  a  small  bacterium,  coccoid  in  appearance.  No  polar  bodies  or 
granules  seen  when  stained  with  the  Neisser  stain!  It  grows  well  in  dextrose, 
lactose,  saccharose,  maltose,  mannite,  dextrin  and  raffinose  broth  and  produces 
acid  in  dextrose  and  maltose.  At  room  temperature  abundant  growth  takes 
place.     Gelatin  is  not  liquefied. 

Hemolysis  of  rabbit  corpuscles  by  diphtheroids  is  not  usually  attributed  to 
any  but  the  true  Klebs-Loeffler  bacterium  from  severe  cases  (Schwoner).  The 
organisms  isolated  from  the  tonsils  were  in  all  cases  but  one,  culturally  unlike 
the  diphtheria  organism  and  in  this  one  instance  proved  to  be  an  avirulent 
diphtheroid,  for  which  the  name  C.  pseudodiphtheriae  sp.  nov.  is  proposed. 
This  organism  grows  on  plain  agar,  is  morphologically  like  the  Klebs-Loeffler 
bacillus.  It  shows  no  growth  anaerobically  after  7  days  and  is  apparently 
strictly  aerobic.  Some  of  the  strains  from  tonsils  required  blood  for  growth 
and  died  off  rapidly  unless  transferred  frequently.  These  organisms  resembled 
B.  influenzae  and  were  hemoglobinophilic. 

Three  strains  were  found  which  fermented  dextrose,  saccharose,  and  maltose 
vigorously  and  formed  slight  acid  in  dextrin.  Raffinose  remained  neutral  or 
faintly  alkaline.  These  diphtheroids  were  isolated  from  glands  in  patients 
with  Hodgkin's  disease.  In  sugar  broths,  growth  was  abundant  with  even 
clouding.  At  room  temperature,  on  ascitic  agar  abundant  growth  occurs.  At 
37  C.  on  the  same  medium  growth  is  abundant  with  a  heavy  creamy  consistency 
and  brownish  tinge.  The  organisms,  small,  ovoid  or  coccoid,  stain  readily  with 
anilin  dyes,  are  gram-positive,  facultative,  nonmotile,  do  not  form  spores,  are 
not  acid-fast.  On  rabbit  blood-agar  plates,  brown  pigment  is  formed  along  the 
streak  growths.  On  Loeffler's  serum,  abundant  white,  glistening  growth  takes 
place.  One  strain,  on  first  isolation,  from  the  gland  on  blood  plates,  exhibited 
a  remarkable  likeness  to  B.  tuberculosis  in  character  of  growth.  The  colonies 
were  dry  and  heaped  up  and  friable.  This  peculiar  property  did  not  persist  in 
later  transplants.  Antiformin  treatment  showed  these  diphtheroids  to  be  non- 
resistant.     The  name  C,  glandulae  sp.  nov.  is  suggested  for  this  species. 


Diphtheroid  Organisms  21 

While  this  study  was  under  waj',  a  paper  by  Torrey  appeared  dealing  with 
bacteria  associated  with  lymph  nodes.  He  tabulated  4  distinct  groups,  which 
when  analyzed  showed  8  different  groups  according  to  fermentation  reactions. 
One  of  the  groups  included  chromogenic  types,  chiefly  orange-red.  This  species 
I  have  named  C.  aurantiacum.  An  anaerobic  diphtheroid  occurring  in  lymph 
nodes  was  described  by  Torrey  and  named  B.  lymphophilus.  It  is  slender^ 
pleomorphic,  gram-positive,  from  0.5  X  1.2-3.2  mikrons.  It  is  not  acid-fast, 
occurs  singly  or  in  pairs,  is  nonmotile ;  stab  cultures  in  0.5%  agar  show  rods 
of  irregular  morphology  and  uneven  staining.  On  Loeffler's  medium,  raised 
white  growth  occurs.  In  dextrose,  high  acid  is  produced,  even  to  the  amount 
of  10  +.  It  is  markedly  aciduric  and  acidophilic.  Glycerol  is  also  fermented, 
but  lactose,  mannite,  inulin  and  dextrin  are  not  attacked.  No  gas  is  produced. 
In  my  studies,  several  strains  were  isolated  which  exhibited  anaerobic  prefer- 
ence for  a  few  generations  only,  but  were  not  like  Torrey's  strain  either  with 
respect  to  anaerobic  requirements  or  unusual  acid-production  in  any  one  carbo- 
hydrate. Torrey's  results  are  of  special  interest  in  showing  that  there  are 
several  types  of  diphtheroids  commonly  present  in  the  lymph  nodes  of  Hodgkin's 
disease,  none  of  which  has  definite  pathogenic  properties  or  can  be  accepted  as 
the  cause  of  the  disease.  In  this  respect  he  confirms  the  findings  of  Bloomfield 
and  myself. 

EYE   DIPHTHEROIDS 

Diphtheroids  isolated  from  the  eye  were  found  to  vary  in  cultural  behavior. 
Three  strains  were  obligate  aerobes  and  produced  only  a  very  slight  amount 
of  acid  in  dextrose  with  0.5  c  c  N/20  acid  maltose.  Seven  other  strains,  facul- 
tative, produced  slight  acid  in  the  same  sugars.  One  strain  grew  slightly,  if 
at  all,  on  agar  and  favored  Loeffler's  serum.  All  of  these  grev/  rather  deli- 
cately on  ascitic  agar  and  serum.  At  room  temperature  growth  was  slow.  The 
organisms   were   large,  clubbed   and    striated.     The   type   C.   xerosis   prevailed. 

NASAL   DIPHTHEROIDS 

Ten  strains  were  isolated  from  nasal  discharges,  mostly  in  persons  with 
cold  in  the  head.  Of  these,  5  strains  were  characterized  by  slight  acid  forma- 
tion in  dextrose  with  marked  acid  in  maltose.  The  remaining  5  were  of  2 
types,  one  which  produced  slight  acid,  the  other  abundant  acid  in  dextrose  and 
maltose.  On  Loeffler's  serum,  growth  was  abundant,  white  and  glistening. 
Stained  with  anilin  dyes,  the  organisms  appeared  short  and  thick  with  larger 
striped  forms  predominating.  They  are  gram-positive,  facultative,  nonmotile, 
do  not  form  spores,  are  not  acid  fast  and  grow  well  in  sugar  broths.  At 
room  temperature  moderate  to  abundant  growth  takes  place.  The  type  C. 
striatum  prevailed. 

DIPHTHEROIDS   FROM    ASCITIC   FLUIDS 

Thirty  specimens  from  cases  of  cirrhosis  and  tuberculosis  were  studied 
anaerobically  and  aerobically  for  diphtheroid  organisms.  No  strict  anaerobes 
were  found.  For  anaerobic  cultures,  Buchner  tubes  (Zinsser's  modification*) 
were  used.  Three  strains  were  isolated.  All  are  gram-positive,  nonmotile,  do 
not  form  spores,  are  not  acid-fast,  are  facultative.  Two  strains  grow  very 
rapidly  at  room  temperature,  a  third  does  not.  One  liquefies  gelatin  and  gives 
rise  to  an  offensive  odor  after  several  days.  In  broths,  the  following  results 
were  noted    (litmus  used  as  indicator). 

•  A  piece  of  absorbent  cotton  is  pressed  down     over    the    pyroKallol    to    prevent    rapid 
mixinR  with  the   NaOH. 


22  Frederick  Eberson 

Strain    Dextrose    Lactose     Saccharose     Maltose     Mannite     Dextrin     Raffinose 
9  +  —  +  +  +  +  _ 

42  —  —  —  —  —  __ 

Strain  43  liquefies  gelatin  and  produces  a  putrid  odor.  This  strain,  prac- 
tically conforms  to  the  description  given  by  Graham  Smith  to  a  liquefying 
diphtheroid  which  he  named  B.  diphtheroides  liquefaciens.  Motility  is  lacking, 
however.  As  was  mentioned  before,  it  does  not  seem  as  if  this  property  is 
peculiar  to  the  group  with  which  we  are  dealing.  The  organism  isolated  from 
ascitic  fluid  is  slender,  curved  and  appears  granular.  It  does  not  form  spores, 
is  facultative,  grows  abundantly  with  confluent  mass  on  agar  at  room  tempera- 
ture and  at  37  C.  and  gives  off  a  decidedly  putrid  odor.  On  Loeffler's  serum 
it  produces  a  heavy  white,  moist  and  glistening  layer.  It  is  suggested  that  the 
name  C.  putidum  sp.  nov.  be  given  to  this  species.  Strain  9  is  characterized 
by  its  wide  range  of  fermenting  power.  It  is  a  very  small  rod,  not  much  larger 
than  B.  influenzae.  On  first  isolation,  club-shaped  forms  predominated.  It 
is  gram-positive,  nonmotile,  does  not  form  spores,  is  facultative,  forms  acid 
in  dextrose,  saccharose,  maltose,  mannite  and  dextrin.  It  grows  abundantly 
at  room  temperature  and  at  37  C.  On  Loeffler's  serum,  abundant  growth  occurs. 
It  does  not  liquefy  gelatin.  C.  ascites  sp.  nov.  is  proposed  as  a  name  for  this 
diphtheroid.  Strain  42  is  a  small  gram-positive  organism,  nonmotile,  forms  no 
spores,  grows  moderately  on  ascitic  agar  at  37  C.  In  the  early  generations  this 
organism  showed  a  decided  preference  for  anaerobic  conditions.  No  growth 
takes  place  at  room  temperature  after  10  days.  It  does  not  liquefy  gelatin 
and  does  not  ferment  sugar  broths.  Grows  best  in  raffinose  broth.  This  seems 
to  be  the  most  selective  medium  for  obtaining  abundant  growth.  All  of  the 
diphtheroids  studied  grew  moderately  well  and  for  the  most  part  abundantly 
in  the  presence  of  this  trisaccharid  when  other  sugars  used  for  fermentation 
studies  showed  little  or  no  growth  other  than  a  sediment.  C.  delicatum  sp.  nov. 
is  suggested  as  the  name  for  this  species   (see  blood  diphtheroids). 

In  the  early  generations  this  organism  showed  a  decided  preference  for 
anaerobic  conditions. 

A  peculiarity  noted  in  the  isolations  from  ascitic  fluids  was  the  fact  that 
the  first  generation  always  had  larger  clubbed  and  knobbed  forms.  In  suc- 
ceeding subcultures  only  very  small  diphtheroids  were  seen. 

DIPHTHEROIDS    IN    BLOOD    CULTURES 

Two  Strains  were  isolated  from  blood  cultures  in  cases  of  pneumonia.  In 
the  first  generation  these  showed  larger  clubbed  forms  and  small  rods.  Growth 
is  delicate.  The  organisms  are  small  gram-positive,  nonmotile,  facultative  and 
produce  very  slight  acid  in  dextrose  or  maltose  but  as  a  rule  leave  all  sugars 
unfermented.  Gelatin  is  not  liquefied  and  growth  at  room  temperature  is 
negative  after  10  days.  On  Loeffler's  serum  moderate  growth  occurs.  These 
diphtheroids  have  been  identified  with  C.  delicatum. 

SKIN    DIPHTHEROIDS 

■  Hine  working  with  34  strains  of  diphtheroids  found  a  group  which  was  asso- 
ciated with  skin  wounds  or  diseased  cavities  opening  on  to  or  near  the  sur- 
face. Strains  of  this  group  were  found  to  acidify  dextrose,  saccharose  usually 
and  less  often  maltose.  The  author  did  not  describe  these  organisms.  That 
diphtheroids  can  be  found  on  the  skin  very  readily  is  well  established.  Judging 
by  the  amazing  number  of  sources  from  which  the  organisms  of  this  group  may 


Diphtheroid  Organisms  23 

be  isolated,  it  seems  highly  probable  that  we  have  to  deal  with  saprophytes 
which  abound  everywhere  like  numerous  species  of  the  Coccaceae  group  and 
which  gain  entrance  into  the  body  through  the  various  openings,  both  normal 
as  well  as  pathologic. 

In  the  course  of  this  study  4  strains  were  isolated  from  an  anal  pus  pocket 
and  from  the  skin  on  the  neck.  Two  of  these  strains  which  will  be  described 
below  are  in  accord  with  those  mentioned  by  Hine ;  the  other  2  differ  sufficiently 
from  these  in  fermentative  and  other  cultural  characters  to  warrant  their 
acceptance  as  other  species. 

(1)  Skin  Diphtheroid:  On  Loeffler's  serum,  abundant  dull  white  growth  is 
produced.  On  agar  delicate  growth  takes  place.  Gelatin  is  not  liquefied.  At 
room  temperature  little  growth  is  observed  after  1  week.  The  organism  is 
gram-positive,  nonspore  forming,  nonmotile,  facultatively  aerobic,  small,  ovoid 
and  stains  readily.  Slight  acid  is  produced  in  dextrose  and  maltose,  the  latter 
being  more  strongly  acidified.  It  is  avirulent.  The  name  C.  epidermidis  sp,  nov. 
is  suggested  for  this  species. 

(2)  Anal  Pus  Pocket:  On  Loeffler's  serum  abundant,  moist  and  glistening 
growth  occurs.  On  agar,  heavy  glistening  growth  takes  place  in  broth,  uniform 
clouding.  Ferments  dextrose  and  maltose  vigorously  and  usually  saccharose. 
It  is  gram-positive,  facultative,  nonmotile,  does  not  liquefy  gelatin,  does  not 
form  spores,  and  grows  abundantly  at  room  temperature.  It  is  avirulent.  To 
this  species  the  name  C.  suppuratum  sp.  nov.  is  given.  Similar  strains  have 
been  observed  by  Trumpp  in  empyema  and  by  Hine  in  a  suppurating  neck  gland. 
The  organism  in  no  way  appears  to  be  associated  with  suppuration  except  as  a 
secondary  invader. 

DIPHTHEROIDS    FROM    BRAIN 

One  strain  was  isolated  from  the  brain  in  a  case  of  meningitis.  The  organ- 
ism is  gram-positive,  ovoid,  or  coccoid  in  appearance,  nonmotile,  nonspore 
forming.  It  grows  very  rapidly  at  room  temperature  and  at  37  C.  on  ordinary 
agar.  On  Loeffler's  serum,  growth  is  white  and  moist.  It  is  avirulent  for 
guinea-pigs  and  for  rabbits  injected  intraperitoneally  and  subdurally.  Vigorous 
acid  production  takes  place  in  dextrose,  lactose  and  maltose.  Growth  in  broth 
is  abundant.  Gelatin  is  not  liquefied.  Ortmann  has  reported  a  diphtheroid 
isolated  from  the  brain  in  a  case  of  meningitis  but  has  given  no  description  of 
the  organism.  It  is  purely  a  secondary  invader.  The  name  C.  cerebralis  sp.  nov. 
is  suggested  for  the  species. 

DIPHTHEROIDS   FROM    THE   APPENDIX 

One  strain  was  obtained  from  this  source.  There  was  slight  suppuration  of 
the  appendix.  The  organism  grows  moderately  on  Loeffler's  serum.  On  agar 
moderate  growth  occurs.  Few  clubbed  and  barred  types  were  observed  in 
the  first  generation  and  in  the  next  few  transplants.  Neisser's  granules  are 
present.  The  organism  is  fairly  large,  nonmotile,  gram-positive,  does  not  pro- 
duce spores  and  does  not  liquefy  gelatin.  At  room  temperature  slight  growth 
occurs.  In  dextrose,  lactose,  saccharose,  maltose  and  dextrin  slight  acid  is 
formed.  Heavy  flaking  with  sediment,  but  no  clouding,  occurs  in  the  broths 
mentioned. 

Hine  has  obtained  a  diphtheroid  from  an  appendix  but  has  not  described  the 
organism.  It  is  a  saprophyte  and  a  secondary  invader.  The  name  C.  floccuiens 
sp.  nov.  is  suggested  for  this  species. 


24 


Frederick  Eberson 


In  all,  diphtheroids  were  obtained  from  21  different  sources  and 
these  fell  into  12  distinct  fermentation  groups,  as  shown  in  Table  3. 
The  greatest  number  of  organisms  comprised  the  group  of  nonfer- 
menters  and  here  is  to  be  noted  the  fact  that  of  13  strains  from  Hodg- 
kin's  disease,  8  came  under  this  head.  However,  little  significance  need 
be  attached  to  this  point,  inasmuch  as  strains  from  Hodgkin's  glands 
fell  into  four  other  groups  besides  this  one.  In  the  same  way,  it  is  to 
be  noted  that  strains  from  the  eye  and  nose  fall  into  4  and  3  different 
groups,  respectively. 

table  3 
Source    and    Fermentative    Reactions   of    Diphtheroids 


Source 


i  Dextrose,  Dextrose, 

Dextrose   Saccha-    Maltose 

rose 


;  Acne  (pustule) 

Appendix 

'  Asciticfluid ' 

Bile I 

'•  Blood 

,  Brain 

Ear 

Eye 

>  Lymph  nodes,  Hodg- 
kin's  

,  Lymph  nodes,  leuke- 
mia  \ 

'■  Lymph  nodes,  pseu- 
doleukemia  

;    Marrow 

,   Nose 

^  Pus  (anal  pocket) 

'  Tonsil 

'  Urine 

I  Horse,    ealf,    sheep 

.       (PreiszNocard  B.) 

1  Unknown 


Total. 


Dextrose,  Dextrose,  Dextrose, 
Lactose,  Saccha-  Saccha- 
Saccha-       rose,         rose. 

rose.       Maltose,  Maltose, 
Maltose     Dextrin    Mannite, 
Dextrin 


1  1      !        1 


Dextrose, 
Saccha-    Dextrose, 
rose.      Maltose, 
Maltose     Dextrin 


Dextrose, 

Dextrose,  Lactose, 

Dextrose,  Maltose,    Saccha-    Nonfer-  To- 

Lactose,    Lactose,       rose,      raenters  tal; 

Maltose     Dextrin     Maltose, 

Mannite, 

Dextrin 


2  (Leu- 

0 

kemia  1) 

1 

1 

1 

5 

10 

3 

2 

3 

1 

2 

3 

28 


It  becomes  evident  that  neither  the  source  nor  the  cultural  charac- 
ters serve  to  distinguish  the  supposed  cause  of  Hodgkin's  disease  from 
numerous  organisms  which  may  be  regarded  as  harmless  saprophytes ; 
also  that  diphtheroid  organisms  comprise  a  greater  number  of  groups 
than  hitherto  supposed. 

There  appears  to  be  a  definite  correlation  between  fermentative 
properties  and  source  with  regard  to  those  organisms  which  were  iso- 
lated from  the  nose  and  throat.  These  are  characterized  by  relative 
inability  to  ferment  carbohydrates.  Acid  formation  in  dextrose 
occurred  but  6  times  of  19;  in  dextrose  and  saccharose,  3  of  19;  in 


Diphtheroid  Organisms  25 

dextrose  and  maltose,  but  1  in  19,  and  of  the  entire  number  of  strains 
more  than  half  failed  to  ferment  any  sugars.  Acid  formation,  there- 
fore, is  limited  in  its  range,  never  exceeding  2  carbohydrates  and  show- 
ing weak  splitting  action  in  general. 

classification 

Classification  from  the  cultural  standpoint  is  difficult  because  of  the 
manifold  variations  which  are  observed.  The  resolution  of  all  diph- 
theroids into  a  few  distinct  "types"  such  as  suggested  by  Morse  is 
possible  only  for  a  limited  number  of  organisms  from  a  limited  number 
of  sources.  Lehmann  and  Neumann  classify  "pseudodiphtheria"  from 
eye  and  nose  as  Coryneb.  pseudodiphtheriticum  and  Coryneb.  xerosis, 
including  diphtheroids  from  both  sources  under  each  heading. 

More  recent  work  such  as  that  of  Plotz  on  typhus  fever  (C.  typhi- 
exanthematici)  indicates  that  the  diphtheroid  group  may  possess  patho- 
genic species  which  are  far  removed  from  the  type  C.  diphtheriae.  The 
organism  described  by  Plotz  and  his  co-workers  is  an  obligate  anaerobe 
with  characteristic  serum  reactions.  Some  diphtheroids  might  be 
classified  from  the  viewpoint  of  human  and  animal  diseases,  and  par- 
ticularly striking  cultural  characters  such  as  oxygen  requirement  and 
sugar  fermentations.  C.  acnes  is  anaerobic  and  might  very  well  go 
into  this  group  of  anaerobes.  Here  also  belong  those  "oxygen  tension" 
diphtheroids  which  are  not  anaerobic  in  the  usual  sense  of  the  word» 
but  which  grow  beneath  the  surface  in  solid  mediums.  The  property 
of  gelatin  liquefaction,  not  hitherto  recognized  as  a  diphtheroid  charac- 
ter, suggests  another  subgroup. 

With  these  points  in  mind  the  following  classification  is  offered.. 
An  effort  has  been  made  to  group  the  species  logically  but  it  is  evident; 
that  relationships  between  different  types  do  not  always  permit  the 
most  desirable  grouping.  Certain  characters  were  used  as  a  basis  for 
arranging  the  distinct  species  under  a  chosen  type.  These  will  become 
evident  as  the  tabulation  is  studied.  In  the  key  given  here,  all  of  the 
known  diphtheroids  have  been  reviewed  and  certain  new  species  added. 

classification  of  diphtheroids 

Group  1. — Diphtheria.  Type  C.  diphtheriae  (Klebs-Loeffler). — 
Nonmotile,  nonsporeforming,  gram-positive,  does  not  liquefy  gelatin, 
shows  polar  bodies  in  young  cultures.  Aerobic  and  grows  between  19 
and  42  C.     Acid  produced  in  dextrose,  maltose  and  dextrin.     Produces 


26  Frederick  Eberson 

toxin  and  gives  rise  to  clinical  symptoms  known  as  diphtheria,  for 
which  a  specific  antitoxin  exists. 

C.  pseudodiphtheriae  sp.  nov.  (Fig.  10).  —  Avirulent  organism, 
morphologically  and  culturally  like  C.  diphtheriae  but  produces  no 
toxin  and  exerts  no  action  when  injected  into  animals.  Isolated  from 
hypertrophied  tonsils. 

Group  2. — Pseudodiphtheriticum.  Type  C.  pseudodiphtheriticum 
(Hofmann-Wellenhoff.) — Nonmotile,  nonsporeforming,  gram-positive, 
does  not  liquefy  gelatin.  It  is  shorter  and  thicker  than  the  C.  diph- 
theriae, but  variations  such  as  knobbed,  clubbed  and  barred  forms 
occur.  It  is  avirulent  and  produces  no  toxin.  In  carbohydrates,  no 
acid  is  produced. 

C.  ceruminis  (Graham  Smith)  Eberson. — Differs  somewhat  from 
C  pseudodiphtheriticum  in  morphology,  as  well  as  in  its  behavior  in 
broth  which  remains  clear,  with  sediment.  In  carbohydrates,  no  acid 
is  produced.     It  is  avirulent.     Isolated  from  normal  ear. 

C.  delicatum  sp.  nov.  (Fig.  6). — Avirulent  strains  from  ascitic 
fluids  and  blood  culture.  The  organism  is  small  and  delicate  with  a 
decided  preference  for  anaerobic  conditions  in  the  early  generations. 
Some  strains  form  traces  of  acid  in  dextrose  or  maltose,  but  usually 
ferment  none  of  the  sugars. 

Group  3. — Xerosis.  Type  C.  xerosis  (Neisser  and  Kuschbert). — 
Nonmotile,  nonsporeforming,  gram-positive,  does  not  liquefy  gelatin. 
Resembles  C.  diphtheriae  closely  morphologically.  Acid  is  produced 
in  dextrose,  saccharose  and  maltose.  The  organism  can  be  isolated 
from  normal  as  well  as  diseased  eyes.  Growth  solid  medium  is  rather 
delicate. 

C.  epidermidis  sp.  nov.  (Fig.  7).  —  Gram-positive,  facultatively 
aerobic,  non-sporeforming,  does  not  liquefy  gelatin.  It  is  small  ovoid 
in  form.  Little  growth  takes  place  after  one  week  at  temperature 
below  25  C.  On  Loeffler's  serum  abundant,  dull  white,  growth.  Slight 
acid  is  produced  in  dextrose  and  maltose.  It  is  avirulent.  Isolated 
from  skin  and  pus  pockets. 

C.  suppuratum  sp.  nov.  (Figs.  3,  4). — Gram-positive,  nonspore- 
forming, does  not  liquefy  gelatin.  Heavy  glistening  growth  on  agar 
and  on  Loeffler's  serum.  Ferments  dextrose  and  maltose  vigorously 
and  usually  saccharose.  Isolated  from  pus  pockets  and  abscesses. 
Avirulent. 


Diphtheroid  Organisms  27 

C.  auris  (Graham  Smith)  Eberson. — Gram-positive,  nonsporeform- 
ing,  does  not  liquefy  gelatin.  Long  barred  or  granular,  often  clubbed. 
Grows  scantily  on  agar.  Acid  in  dextrose  and  maltose,  but  not  saccha- 
rose.    It  is  often  virulent  for  guinea-pigs.     Isolated  from  ear. 

C.  cerebralis  sp.  nov.  (Fig.  11). — Gram-positive,  ovoid  or  coccoid, 
honmotile,  nonsporeforming,  does  not  liquefy  gelatin.  Grows  rapidly 
at  room  temperature  and  at  37  C.  on  agar.  On  Loeflfler's  serum,  growth 
is  white  and  moist.  Vigorous  acid  production  in  dextrose,  lactose  and 
maltose.     Avirulent  for  guinea-pigs  and  rabbits.     Isolated  from  brain. 

Group  4. — Nodosum.  Type  C.  nodosum  (Migula)  Eberson. — 
Large,  clubbed  forms  markedly  segmented.  Does  not  form  spores,  is 
gram-positive,  nonmotile,  does  not  liquefy  gelatin,  white  growth  on 
agar.  Acid  in  dextrose,  saccharose,  maltose,  and  dextrin.  Found  fre- 
quently in  urine  and  the  urogenital  tract.     Avirulent. 

C.  acidum  sp.  nov.  (Figs.  1,  2). — Gram-positive,  nonmotile,  small 
rods,  with  abundant  white,  glistening  growth  on  agar.  No  spores,  is 
aerobic  and  does  not  liquefy  gelatin.  Produces  large  amounts  of  acid 
in  dextrose,  lactose,  saccharose,  maltose,  mannite  and  dextrin,  but  does 
not  attack  raffinose.    Isolated  from  urine.    Avirulent. 

C.  ascites  sp,  nov.  (Fig.  8). — Gram-positive,  small  rod  with  numer- 
ous club-shaped  forms  on  first  isolation.  Nonmotile,  nonsporeforming, 
does  not  liquefy  gelatin,  grows  abundantly  on  agar  and  Loefifler's 
serum.  Forms  acid  in  dextrose,  saccharose,  maltose,  mannite  and 
dextrin.    Avirulent.    Isolated  from  ascitic  fluid. 

C.  flocculens  sp,  nov.  (Fig.  13). — Gram-positive,  nonmotile,  clubbed 
or  barred  forms,  does  not  form  spores  or  liquefy  gelatin.  The  organ- 
ism grows  moderately  on  agar  and  Loeffler's  serum  at  37  C.  At  room 
temperature  slight  growth.  Neisser's  granules  present.  Produces  acid 
in  dextrose,  lactose,  saccharose,  maltose  and  dextrin.  Heavy  flaking 
with  sediment  without  clouding  in  broths.  Isolated  from  a  suppurat- 
ing appendix. 

Group  5.  —  Pigment- forming  Diphtheroids.  Type  C.  hoagii 
(Morse)  Eberson. — Medium  sized  bacterium  with  solid,  barred  and 
wedge-shaped  forms  with  abundant  but  imperfect  granules.  On  serum 
a  heavy,  confluent,  glistening  growth  with  a  characteristic,  salmon-pink 
color.  Gram-positive,  forms  no  spores,  does  not  liquefy  gelatin,  non- 
motile.    Produces  acid  in  dextrose  and  saccharose,  but  not  in  maltose. 

C.  aurantiacum  sp.  nov.  (Fig.  5), — Gram-positive,  small  and  coc- 
coid, nonmotile,  nonsporeforming,  does  not  liquefy  gelatin,  not  acid- 


28  Frederick  Eberson 

fast.  Grows  abundantly  on  agar  at  room  temperature  and  at  37  C. 
and  produces  a  bright  orange  pigment.  No  polar  bodies  or  granules. 
Grows  well  in  sugar  broths  and  produces  acid  in  dextrose  and  maltose. 
Pigment  production  in  24  hours  under  aerobic  and  anaerobic  condi- 
tions at  18-25  C.,  but  at  37  C.  pigment  in  this  length  of  time  only  under 
aerobic  conditions.  Under  anaerobic  conditions,  at  37  C.  a  trace  of 
pigment  was  evident  after  10-14  daysi. 

C.  glandulae  sp.  nov.  (Fig.  9). — Small  ovoid  or  coccoid  bacterium. 
Grows  abundantly  at  room  temperature  or  at  37  C.  with  a  heavy  creamy 
consistency  and  brownish  tinge.  Gram-positive,  nonmotile,  nonspore 
forming,  does  not  liquefy  gelatin.  Grows  abundantly  in  sugar  broths 
with  uniform  clouding.  On  LoefHer's  serum  abundant  white  glistening 
growth  occurs,  changing  to  a  brownish  color  after  a  few  days.  Pro- 
duces acid  in  dextrose,  saccharose  and  maltose  vigorously  and  slight 
acid  in  dextrin.  RafSnose  neutral.  One  strain  on  first  isolation  from 
the  gland  on  blood  plates  showed  a  remarkable  likeness  to  B.  tubercu- 
losis in  character  of  growth ;  the  colonies  were  dry  and  heaped  up  and 
friable.  This  peculiarity  did  not  persist  in  later  transplants.  Anti- 
formin  treatment  showed  these  diphtheroids  to  be  nonresistant.  They 
were  not  acid-fast.     Isolated  from  glands  of  Hodgkin's  disease. 

C.  striatum  (Chester)  Eberson. — Thick  form,  with  clear  cut  bars. 
Large  and  irregular  granules  visible.  Heavy  growth  on  serum  with 
pigment  from  white  to  yellow.  Ferments  dextrose  and  maltose,  but 
not  saccharose.  Gram-positive,  does  not  form  spores,  nonmotile,  does 
not  liquefy  gelatin.     Found  in  normal  nasal  mucus. 

C.  segmentosum  nom.  nov. — Nonmotile,  gram-positive,  nonspore- 
forming,  does  not  liquefy  gelatin.  Polar  bodies  demonstrable.  In 
broth,  growth  is  clear  with  sediment.  Slight  acid  is  produced  in  dex- 
trose.   Avirulent,  from  nasal  mucus. 

Group  6. — Gelatin-liquefiers.  Type  C.  putidum  sp.  nov.  (Fig.  12). 
— A  slender,  curved  granular  bacterium.  It  produces  no  spores,  is 
facultative,  grows  abundantly  with  confluent  stringy  mass  on  agar  at 
room  temperature  and  at  37  C.,  giving  ofif  a  decidedly  offensive  odor. 
On  Loeffler's  serum,  a  heavy,  white,  moist  and  glistening  mass.  It 
forms  no  acid  in  dextrose,  lactose,  saccharose,  maltose,  mannite,  dex- 
trin and  raf¥inose.     Isolated  from  ascitic  fluid. 

Group  7. — Anaerobic  Diphtheroids  Associated  with  Specific  Clin- 
ical Manifestations.  Type  C.  typhi-exanthematici  (Plotz). — Small, 
pleomorphic,  gram-positive  organism,  not  motile,  not  acid-fast.     Most 


Diphtheroid  Organisms  29 

of  the  bacteria  are  straight,  some  slightly  curved,  coccoid  forms  also 
occur.  The  ends  are  slightly  rounded  or  pointed.  Nonspore  forming. 
It  is  an  obligate  anaerobe  and  can  be  isolated  on  special  glucose  medi- 
ums containing  ascitic  fluid  of  a  definite  specific  gravity.  Produces  acid 
in  dextrose,  galactose,  maltose  and  inulin.  Found  in  typhus  fever  with 
regularity ;  specific  serum  reactions  ;  pathogenic. 

C.  acnes  (Gilchrist)  Eberson. — Gram-positive,  small  organism, 
resembling  C.  typhi-exanthematici.  Stains  unevenly ;  anaerobic,  but 
many  strains  grow  aerobically  after  continued  cultivation.  Surface 
smears  fail  to  show  growth  with  material  from  pustules,  whereas  deep 
stab  cultures  and  heavily  inoculated  unbroken  pustules  always  give 
growth.  Mediums  with  large  amounts  of  acid  especially  adapted  for 
isolating  this  organism  which  produces  acid  in  dextrose,  saccharose 
(slight),  maltose,  mannite  and  inulin.  Raffinose  is  fermented  by  some 
strains.     Pathogenic  for  mice  and  gives  rise  to  cliaracteristic  lesions. 

Group  8. — Pyogenic  Diphtheroids  in  Animals.  Type  C.  pseudotu- 
berculosis (Preisz)  Eberson. — Gram-positive,  nonmotile,  club-shaped. 
On  serum  heavy  moist  growth  with  golden-yellow  pigment.  Toxin 
production  is  attributed  to  the  organism  and  it  is  fatal  for  guinea-pigs, 
rabbits,  goats  and  sheep.  Blood  agar  with  a  minimum  amount  of  fer- 
mentable carbohydrate  shows  hemolysis.  Acid  in  dextrose  and  maltose, 
but  not  glycerin.    Produces  suppurative  processes  in  the  lymphatics. 

C.  pyogenes  (Glage). — Gram-positive,  nonmotile,  club-shaped  or 
cylindrical.  Does  not  grow  at  room  temperature.  Is  facultatively 
aerobic,  produces  no  spores  and  is  hemoglobinophilic.  Pathogenicity 
doubtful.     No  acid  produced  in  dextrose  or  glycerol. 

C.  vaccinae  (Galli-Valerio).  —  Gram-positive,  nonmotile,  club- 
shaped,  coccoid,  segmented  or  branched.  Facultatively  anaerobic,  non- 
spore  forming.  On  serum,  growth  abundant;  pale  yellow  or  orange- 
yellow  pigment  produced ;  gelatin  not  liquefied.  Pathogenic  for  guinea- 
pigs,  mice  and  rabbits.     Associated  with  vaccine  pustules  in  calves. 

Group  9. — Anaerobic  Diphtheroids  Associated  with  Diseased 
Lymph  Glands.  Type  C.  lymphophilus  (Torrey). — Slender,  pleomor- 
phic, gram-positive,  nonmotile ;  markedly  aciduric  and  acidophilic ;  fer- 
ments dextrose  and  glycerol;  avirulent.  From  lymph  nod€S  in 
Hodgkin's  disease. 

DISCUSSION 

From  this  study,  which  is  far  from  complete,  it  is  evident  that  the 
sources  of  diphtheroid  organisms  have  not  yet  been  exhausted.  That 
many  strains  are  yet  to  be  described  is  a  foregone  conclusion.     The 


30  Frederick  Eberson    . 

bacteriologc  study  of  body  fluids  and  glands  and  of  secondary  invaders 
in  common  diseases  will  undoubtedly  give  us  material  for  ultimate 
classification.  Apart  from  Morse's  biometric  study  of  the  diphtheria 
group,  no  other  classification  has  been  presented.  In  the  light  of  the 
material  given  here  it  seems  as  if  Morse's  classification  on  the  basis  of 
chromogenesis,  vigor  of  growth  and  fermentative  properties  is  not 
entirely  adapted  to  the  great  number  of  strains  which  have  already  been 
described  and  which  are  forthcoming.  Serologic  studies  as  a  means  of 
classification  in  this  group  of  bacteria  have  not  been  worked  out  suffi- 
ciently to  merit  much  discussion;  the  little  work  done  has  not  been  of  a 
very  searching  nature.  It  seems  that  here  are  the  most  prom.ising 
possibilities.  Agglutination  has  been  tried  by  Nicholas,  Nicolle,  Land- 
steiner,  Bruno,  Lubowski,  Lesieur,  and  Fraenkel  with  varying  results, 
which  were  not  specific.  The  consensus  of  opinion  seems  to  be  that 
the  organisms  do  not  produce  agglutinating  substances  in  the  serum. 
Complement  fixation  has  not  been  sufficiently  studied.  The  results  of 
Morse  show  that  cross-reactions  m.ay  be  obtained  for  the  diphtheroid 
antigens  and  that  C.  diphtheriae  does  not  fix  with  any  of  the  diph- 
theroid antigens.  The  results,  however,  are  not  striking  enough  to  be 
conclusive.  The  method  of  preparing  antigen  undoubtedly  will  modify 
the  results  in  such  tests.  Ground  bacteria  were  used  and  specific  serum 
obtained  by  the  injection  of  formalized  suspensions  of  whole  organ- 
isms. Perhaps  Olitzky's  method  for  complement  fixation  in  typhus 
fever,  would  be  preferable.  According  to  his  technic,  nonspecific  fixa- 
tion is  prevented  by  the  use  of  Berkefeld  filtrates  of  autolyzed  bacteria. 
It  is  hoped  that  by  means  of  group  reactions  the  diphtheroids  may  be 
classified  definitely.  Precipitation  tests  suggest  good  possibilities.  The 
reaction  is  exceedingly  delicate  and  the  organisms  lend  themselves  to 
ready  disintegration  and  extraction. 


II.    SEROLOGICAL 

experiments   with    hodgkin^s   strains 

In  order  to  determine  the  identity  of  cultures  isolated  from  cases  of 
Hodgkin's  disease,  two  methods  were  utilized — agglutination  and  com- 
plement fixation.  The  first  method  enables  us  to  identify  the  specifi- 
city of  the  organisms  by  means  of  serums  obtained  from  patients  suf- 
fering with  the  disease  as  well  as  by  serums  prepared  with  the  dififerent 
strains  of  bacteria  isolated.     Obviously,  if  the  C.  Hodgkinii  is  the  cause 


Diphtheroid  Organisms  31 

of  Hodgkin's  disease  we  ought  to  be  able  to  show  a  definite  relationship 
between  serums  from  patients  and  organisms  isolated  from  these  and 
other  cases.  By  the  delicate  method  of  complement  fixation  we  may 
detect  similar  antigenic  substances  and  establish  the  identity,  if  such 
there  be,  of  the  several  strains  isolated. 

Agglutination 

Four  serums,  obtained  from  4  authentic  cases  of  Hodgin's  disease  were 
studied.  Eleven  strains  of  bacteria  were  tested,  of  which  8  were  isolated  from 
glands  in  Hodgin's  disease  and  the  remaining  3  from  a  case  of  leukemia, 
ascitic  fluid  in  cirrhosis  and   blood. 

Forty-eight-hour  agar  cultures  were  suspended  in  salt  solution  and  emul- 
sions made  up  to  approximately  the  same  density.  These  were  shaken  with 
glass  beads  for  thirty  minutes  and  then  centrifugated  for  5  minutes  at  low 
speed  to  throw  down  coarser  particles.  Equal  volumes  of  the  different  serums 
(diluted  with  NaCl  solution)  were  mixed  with  the  bacterial  suspensions  in  a 
hanging  drop.  The  preparations  were  well  sealed  with  vaselin  and  incubated  at 
room  temperature  for  1  hour,  then  at  38  C.  for  another  hour,  when  the  results 
were  read. 

The  results  were  entirely  negative  and  confirm  the  results  of  Fox. 

Complement  Fixation 

In  these  experiments  the  aim  was  to  show  by  tests  for  cross  fixation  what 
relationship,  if  any,  existed  between  the  different  types  of  bacteria  isolated 
from  cases  of  Hodgkin's  disease.  The  cultures,  as  in  the  preceding,  were 
obtained  from  Drs.  Bunting,  Yates  and  Rosenow  and  from  cases  studied  by 
me.     These  strains  have  been  described   fully  in  Part  I. 

Four  strains  were  used  in  the  production  of  immune  serums  from  rabbits. 
These  cultures  are  designated  2,  8,  13  and  57.  No.  2  was  isolated  by  Dr.  C.  H. 
Bunting  from  a  patient  who  had  a  very  acute  form  of  Hodgkin's  disease ;  it 
fermented  dextrose  and  saccharose  with  acid.  No.  8  was  sent  to  me  by  Dr. 
E.  C.  Rosenow,  and  labeled  B.  hodgkinii;  it  produced  acid  in  dextrose,  maltose, 
saccharose,  and  dextrin.  No.  13  was  obtained  from  the  same  source;  it  fer- 
mented dextrose  and  maltose.  No.  57  was  isolated  by  me  from  a  case  of  acute 
Hodgkin's  disease.  This  organism  fermented  none  of  the  carbohydrates  and 
was  identical  in  this  respect  with  a  strain  (No.  1)  obtained  from  Dr.  Bunting. 

Healthy  rabbits,  weighing  about  1,600  gm.,  were  systematically  treated  with 
each  of  these  cultures  heated  to  58  C.  for  30  minutes.  Injections  were  made 
with  increasing  doses  every  week  for  a  period  of  6  weeks  and  the  serums  drawn 
10  days  after  the  last  injection. 

Antigens  were  prepared  by  grinding  to  a  fine  powder  the  flocculent  precipi- 
tate obtained  from  a  mixture  of  bacteria  with  an  excess  of  absolute  alcohol. 
The  powder  was  dried  in  vacuo  and  then  ground  in  a  mortar  with  solid  NaCl. 
Subsequently  sterile  distilled  water  was  added  to  isotonicity  and  the  anti- 
complementary unit  determined. 

Complement  was  obtained  from  normal  guinea-pigs  and  titrated  in  the 
usual  way.  Sensitizer  was  prepared  with  thoroughly  washed  sheep  blood  cells 
injected  into  rabbits  and  the  unit  determined,  after  which  a  5%  suspension 
of  sheep  cells  was  sensitized  with  2  units  of  the  amboceptor.  Readings  were 
taken  after  the  tubes  were  incubated  for  15  minutes  in  a  i7  C.  water  bath,  and 
8  hours  in  the  ice-chest. 


32 


Frederick  Eberson 


The  results  showed  no  correspondence  among  the  separate  strains  and  hence 
prove  them  to  be  nonidentical,  a  fact  which  confirms  the  cultural  differences 
observed  in  the  same  organisms.  Fox  failed  to  obtain  fixation  with  the  serum 
of  a  patient  and  a  strain  isolated  from  the  glands. 


TABLE  1 
Relationship    of   Type   Diphtheroids 


Serum  Antl-hoagil 


Absorbed  with 

Before  Absorption 

Pseudodiphtlieriae 

Plavidus  (Mellon) 

Dilution 

Pseudo- 

flavidus 

Pseudo- 

Flavidus 

Hoagii           diph- 

(MeUon 

Hoagii 

diph- 

Hoagii 

(Mellon) 

theriae 

Strain) 

theriae 

1-20 

1     ++     :      + 

+  + 

+  + 

_ 

++ 

— 

1-40 

I     ++     i      + 

+  + 

++ 

— 

++ 

— 

1-80 

++          + 

+ 

+  + 

— 

+  + 

— 

1-160 

++          ± 

+ 

+  + 

— 

++ 

— 

1-320 

++ 

+ 

+  + 

— 

+  + 

— 

1-500 

++ 

■+■ 

++ 

++ 

1-1,000 

+ 

— 

+ 

■+■ 

1-1,500 

+      1 

± 

— 

1-2,000 

±      ! 

— 

~ 

Serum  Anti-pseudodiphtheriae 


Before  Absorption 

Absorbed  with  Flavidus  (Mellon) 

Dilution 

Pseudo- 

Flavidus 

Hoagii 

Pseudo- 

Flavidus 

diphtheriae 

(Mellon) 

diphtheriae 

(Mellon) 

1-20 

+  + 

+ 

+ 

++ 



1-40 

+  + 

+, 

+ 

++ 

— 

1-80 

+  + 

+ 

-h 

+  + 

— 

1-160 

+  + 

-+- 

— 

+  + 

— 

1-320 

+  + 

— 

+ 

1-500 

+  + 

— 

■+■ 

1-1,000 

+    ■ 

— 

1-1,200 

Serum  Antiflavidus  (MeUon) 


Before  Absorption 


Absorbed  with  Flavidus  (Morse) 


Dilution 

Flavidus 

Pseudo- 

Flavidus 

Flavidus 

(Mellon) 

diphtheriae 

(Mellon) 

(Morse) 

1-20 

+ 

±                        ': 

+ 

_ 

1-40 

+ 

—                        i 

4- 

— 

1-80 

+ 

—                        1 

+ 

— 

1-160 

+ 

— 

+ 

— 

1-320 

+ 

—                             ; 

+ 

— 

1-500 

+ 

~" 

+ 

" 

AGGLUTININ    ABSORPTION    TO    DIFFERENTIATE    TYPE    DIPHTHEROIDS 

The  literature  on  diphtheroids,  to  my  knowledge,  does  not  contain 
any  reference  to  agglutinin  absorption  as  a  method  for  showing  rela- 
tionships which  might  exist  among  overlapping,  dosely-related  types. 
In  view  of  the  fact  that  the  differences  which  characterize  the  accepted 


Diphtheroid  Organisms  33 

forms  are  so  slight,  bacteriologically  speaking,  such  a  method  suggested 
possibilities  for  more  definite  classification. 

The  strains  studied  were  obtained  from  the  Museum  of  Natural  History  and 
were  marked  as  follows :  C.  hoagii,  C.  flavidus  (Mellon),  C.  pseudodiphtheriae, 
C.  xerosis,  C.  enzymicus  (Mellon),  and  C.  flavidus  (Morse). 

Agglutinating  serums  were  prepared  in  rabbits  by  giving  intravenous  injec- 
tions of  fresh  agar  cultures  heated  at  56  C.  for  30  minutes.  Increasing  doses 
were  given  every  week  until  a  whole  culture  was  reached.  The  animals  gained 
weight  throughout  treatment  and  were  bled  10  days  after  the  last  injection. 
Preliminary  titrations  were  made  with  each  of  the  serums  against  homologous 
and  heterologous  strains,  and  double  absorption  of  each  serum  was  made  with 
organisms  which  evidenced  some  affinity  for  the  agglutinins  present. 

The  results  of  the  absorption  experiments  indicate  that  C.  hoagii  is 
more  inclusive  in  its  antigenic  elements,  since  it  possesses  group  agglu- 
tinins for  all  the  other  strains,  excepting  C.  xerosis.     C.  flavidus  (Mel- 

TABLE  2 
C0.WPARATIVE   Results 

Flavidus  (Morse)        Flavidus  (Mellon) 

Dextrose 3.3  cc  2.0  c  c 

Maltose 2.0  1.7 

Saccharose 2.5  i  1.4 

Glycerol 3.3  2.6 

The  figures  represent  the  amount  of  N/20  NaOH  required  to  neutralize  5  c  e  ol  broth 
culture  incubated  1  day  at  37  C.  Growth  was  heavy  in  both  sets.  The  Mellon  strain  gave 
a  flocculate  growth  and  the  Morse  culture  was  uniformly  clouded. 

Ion)  has  no  group  agglutinins  for  any  of  the  others  and  appears  to  be 
highly  specific.  The  results  vary  somewhat  as  compared  with  Morse's 
complement  fixation  results.  Mellon's  flavidus  shows  no  group  agglu- 
tinins at  all,  although  Morse  obtained  partial  cross  fixation  with  her 
strain.  It  appeared  likely  that  the  former  culture  sent  to  the  Museum 
of  Natural  History  as  a  **B.  flavidus,"  isolated  by  Mellon,  was  not  a 
flavidus  type.  When  its  homologous  serum  was  absorbed  with  C. 
"flavidus"  (Morse),  the  specific  agglutinins  remained  unchanged.  That 
complete  absorption  by  the  heterologous  strain  took  place  is  evident 
from  the  table.  Macroscopically,  the  Morse  culture  differs  markedly 
on  plain  agar  from  the  Mellon  strain.  The  latter  develops  a  dull,  dry 
growth,  with  a  tendency  to  flocculate  readily  in  NaCl  solution,  whereas 
the  former  is  moist  and  glistening  in  its  growth.  In  carbohydrates  the 
comparative  results  were  as  shown  in  Table  2. 

The  relative  rate  of  acid-production  by  both  strains  is  obviously 
different.  Prolonged  cultivation  may  give  somewhat  different  results, 
but  it  has  been  shown  bv  several  workers  that  the  maximum  acid- 


34  Frederick  Eberson 

production  in  the  usual  carbohydrate  concentrations  is  at  a  maximum 
after  24  hours.  In  any  case  the  antigenic  properties  of  both  strains  are 
not  in  agreement,  and  the  cultural  tests  seem  to  bear  out  this  difiference. 
Mellon's  flavidus  may  be  a  subspecies,  but  it  differs  from  Morse's  tyoe 
too  markedly  to  go  by  the  same  species  name. 

Closer  agreement  exists  in  the  case  of  C.  hoagii,  for  which  we  find 
on  the  one  hand  neither  group  agglutinins  nor  cross-fixation  for  C. 
hofmanni  and  C.  xerosis,  while,  on  the  other  hand,  there  are  both 
cross-fixation  and  group-agglutination  with  the  flavidus  antigen.  C. 
hofmanni  shows  group  agglutinins  for  C.  hoagii  and  C.  flavidus  (Mel- 
lon), but  not  for  C.  xerosis. 


Agglutination   Tests 

TABLE  3 

AND   Complement 

Fixation    Study 

Antiserum 

Antigen 

C.  flavidus 

C.  hoagii ,       !        C.  xerosis 

C.  hofmanni 

C.  flavidus 

C.  hoagii 

0.025    (500) 

0.05      (600) 

(160) 

1      0.1       (  -  ) 

0.025    (2000) 

(80) 

0.1    (  -  ) 
...     (-) 

0.4    (  20  ) 
...     (  160) 

C.  hofmanni 

(1000) 

The  figures  and  signs  enclosed  in  parentheses  represent  the  highest  dilution  in  which 
partial  agglutination  occurred,  and  the  decimals  indicate  the  amount  of  antigen  in  mg.  which 
was  necessary  for  fixation  with  the  difTerent  serums.  Absorption  experiments  with  a  strain 
of  Morse  flavidus  omitted. 

There  is  no  apparent  correlation  between  the  reactions  in  carbo- 
hydrates and  the  antigenic  properties.  Nonspecific  agglutinins  appear 
to  be  readily  absorbed,  whereas  the  specific  agglutinins  are  left  intact. 

Morse's  complement  fixation  study  and  the  group  agglutination  tests 
are  shown  in  Table  3. 


III.     PLEOMORPHISM     OF     DIPHTHEROIDS,     MUTABILITY 

OF    TYPES,    AND    A    METHOD    FOR    DETERMINING 

MUTANTS 

VARIABILITY    OF     BACTERIA 

Bacteria  are  unicellular  organisms  which  are  produced  from  other 
like  organisms  by  the  process  of  fission — an  act  in  which  the  entire 
parent  substance  may  be  said  to  divide  itself  equally  into  two  fractions, 
both  of  which  constitute  the  resulting  pair  of  bacteria.  Under  such 
conditions  the  offspring  cannot  possess  what  was  not  present  in  the 
parent.  This,  in  essence,  is  the  pure  line  concept  of  bacteria  and 
implies  the  descendants  from  any  single  bacterial  cell.  The  hereditary 
charaqteristics  of  such  a  biotype  do  not  change  as  a  result  of  changed 


Diphtheroid  Organisms  35 

environmental  conditions.  In  order  to  explain  variations  in  bacterial 
types  it  is  necessary  to  consider  both  Darwin's  theory  and  that  of 
deVries.  The  former  conception  is  based  on  natural  selection  in  which 
such  variations  as  are  better  adapted  to  the  struggle  for  existence  will 
be  perpetuated.  The  theory  of  deVries  holds  that  the  tendency  toward 
variation  in  the  germ  plasm  may  give  rise  to  permanent  variations  as 
opposed  to  fluctuating  variations. 

When  bacteriology  was  in  its  infancy  it  was  a  popular  notion  that 
types  could  be  transmuted  readily  from  one  into  the  other  and  back 
again.  Strange  as  it  may  seem  the  idea  has  been  reborn  and  given 
great  impetus  within  recent  years.  Pleomorphism  is  highly  developed 
among  the  diphtheroids  and  it  is  in  this  group  that  much  confusion  has 
arisen  not  only  as  a  natural  result  of  the  well  known  overlapping  of 
closely  related  types,  but  chiefly  because  of  the  ease  with  which  appar- 
ently pure  cultures  of  the  organisms  undergo  changes  in  their  mor- 
phology. In  none  of  the  work  presented  has  the  technic  been  of  such  a 
nature  as  to  preclude  error.  Billings  and  Rosenow  state  that  single 
colonies  of  diphtheroids  from  Hodgkin's  disease  "in  dextrose  agar 
which  showed  bacilli  only  in  smears,  yielded  in  subcultures  a  pure 
culture  of  staphylococci  aerobically  and  forms  of  the  bacillus  either 
pure  or  in  mixture  anaerobically  on  the  same  medium.  These  facts 
suggest  strongly  that  the  associated  staphylococcus  is  derived  from  the 
bacillus."  Certainly,  no  evidence  is  given  by  the  authors  that  the 
single  colonies  were  pure.  There  is  only  one  method  whereby  the 
fallacy  may  be  precluded  and  that  method  was  not  used  in  this  par- 
ticular instance.  It  may  or  may  not  be  true  that  the  staphylococcus 
was  associated  with  the  bacillus,  but  the  word  "derived"  is  misleading. 
We  know  from  common  practice  that  a  single  colony  even  when 
obtained  from  highly  diluted  plate  cultures,  does  not  necessarily  repre- 
sent the  descendants  of  a  single  bacterial  cell.  Such  a  mistake  was 
made  by  Goodman  when  he  claimed  to  have  separated  the  diphtheria 
organism  into  an  acid-producing  and  an  alkali-producing  type  by  the 
plate  method,  on  the  assumption  that  he  had  a  pure  line. 

I  shall  deal  exclusively  with  C.  enzymicus  described  and  named  by 
Mellon.  This  organism  exhibits  the  pleomorphism  of  most  diph- 
theroids but  is  of  especial  interest  because  of  the  remarkable  morpho- 
logical changes  the  author  claims  to  have  produced.  He  was  able  to 
show  a  seeming  relationship  between  the  diphtheroids  and  the  strepto- 
cocci and  described  a  method  whereby  he  transformed  the  bacillary  type 
of  diphtheroid  into  a  coccus  and  back  again.     The  old  question  of  sta- 


36  Feederick  Eberson 

bility  of  bacterial  type  comes  up  and  it  is  essential  that  the  cardinal 
points  be  investigated.  Two  methods  of  attack  were  followed  in  my 
studies  with  C.  enzymicus :  cultural  and  serological. 

Cultural   Study   of   Mutation 

Two  strains  of  C.  enzymicus  were  studied.  One  was  obtained  from  the 
Museum  of  Natural  History  and  had  been  cultivated  on  plain  agar  for  several 
months.  Smears  stained  with  Loeflfler's  methylene  blue  showed  typical  diph- 
theroid bacillary  forms  with  the  usual  coccoidal  or  short  rod  and  extremely 
small  forms  (Fig.  14).  The  second  culture  was  sent  by  Dr.  Mellon.  This  strain 
had  been  cultivated  on  blood  agar  for  about  1  year  and  in  smear  preparations 
appeared  to  be  practically  free  from  coccoidal  forms,  showing  large  unevenly 
staining  bacillary  types,  varying  in  size  and  morphology. 

Museum  Strain  of  C.  Enzymicus. — A  subculture  was  made  on  plain  agar 
slants  and  the  24-hour  growth  examined.  Stains  were  made  from  dififerent 
areas  and  examined  with  a  ^2  immersion,  using  a  magnification  of  approxi- 
mately 1,400.  In  all  instances  diplococci  and  coccilike  organisms  were  seen 
grouped  like  staphylococci  and  at  times  singly.  Careful  examination  showed 
clusters  of  bacillary  forms  in  small  numbers   (Fig.  15). 

The  next  step  was  to  obtain  a  pure  culture  of  the  coccus  type.  Agar  plates 
(meat  infusion  agar  1.0  to  phenolphthalein)  were  streaked  with  a  platinum 
loop  touched  to  opiginal  culture  and  several  discrete  colonies  fished.  In  all 
cases  a  single  colony  was  used  to  prepare  a  second  plate  culture  and  from  this 
generation  of  single  colonies,  other  plates  streaked  successively  until  the  10th 
generation.  Each  generation  was  studied  for  morphology  before  seeding. 
Pure  coccus  forms  were  obtained  in  every  case  (Fig.  16).  No  difficulty  what- 
soever was  experienced  in  procuring  a  coccus  form  from  the  original  culture. 
The  real  hardship  lay  in  trying  to  obtain  the  bacillary  type  in  relatively  pure 
culture,   free  from  cocci. 

It  is  a  well  known  fact  that  blood  and  serum  mediums  exert  a  favorable 
influence  on  the  diphtheroid  bacteria,  causing  the  development  of  large  clubbed 
and  barred  forms.  Taking  advantage  of  this  fact,  and  the  fortunate  "trans- 
formation" eflfected  on  plain  agar,  it  becomes  possible  to  demonstrate  the 
mechanism  of  variability  in  diphtheroids. 

Again  using  the  original  culture  of  C.  enzymicus  I  transplanted  it  to  sheep 
blood  agar  and  examined  the  culture  after  24  hours'  incubation.  Smears 
showed  cocci  and  numerous  bacillary  forms.  When  subcultured  on  the  same 
medium  the  bacillary  forms  had  almost  completely  outgrown  the  cocci  and  a 
typical  diphtheroid  picture  was  seen.  As  generation  after  generation  on  this 
medium  was  studied,  it  was  found  that  cocci  disappeared,  although  not  entirely. 
At  times  it  required  careful  examination  of  the  microscopic  field  to  discern 
them  among  the  larger  barred  and  granular  forms.  A  most  interesting  point 
was  next  observed.  When  subculturing  on  sheep  blood,  transfers  were  made  to 
plain  agar  slants  from  the  first  generation  on  sheep  blood  medium.  After 
24  hours'  incubation  the  agar  tubes  were  found  to  contain  cocci  only.  As 
progressive  generations  on  blood  medium  were  transferred  to  plain  agar  it 
became  more  and  more  difficult  to  suppress  the  bacillary  types  and  to  demon- 
strate the  coccus.  By  the  use  of  various  mediums  and  numerous  cross  sub- 
inoculations  it  appeared  quite  clear  that  the  selection  of  biotypes  was  dependent 
on  the  environment.  These  details  are  shown  in  Chart  1.  Certain  cardinal 
points  may  be  observed  by  studying  the  diagram  in  which  evidence  is  adduced 


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38  Frederick  Eberson 

that  we  are  dealing  with  distinct  biotypes  side  by  side  in  a  cuhure  of  bacteria. 
When  conditions  are  optimum  for  a  particular  biotype,  variations  do  not  occur 
readily.  A  new  type  which  may  have  been  brought  out  in  company  with  the 
predominating  type  by  a  change  to  a  less  favorable  environment  can  be  sup- 
pressed as  conditions  are  restored  to  normal.  It  is  to  be  noted,  however,  that 
prolonged  cultivation  under  what  were  at  the  outset  unfavorable  conditions, 
often,  if  not  as  a  rule,  leads  to  adaptation,  and  it  becomes  impossible  to  obtain 
a  single  biotype. 

This  hypothesis  is  supported  strongly  by  the  following  observation :  A  sub- 
culture on  plain  agar  made  from  the  4th  generation  on  sheep  blood  medium 
(bacillary  forms  predominating)  developed  a  peculiar  surface  growth  of  very 
heavy  opaque  masses  lying  in  a  more  or  less  translucent  delicate  growth  which 
composed  the  greater  part  of  the  transplant.  Microscopic  examination  of  the 
heavy  growth  showed  purely  bacillary  forms  with  isolated  cocci,  whereas  the 
translucent  area  gave  cocci  only.  Touching  a  platinum  needle  lightly  to  the 
heavy  mass  a  second  tube  of  plain  agar  Avas  inoculated  and  exactly  the  same 
picture  was  obtained  (Figs.  17,  18,  and  19,  etc.).  This  was  observed  on  blood 
medium  also  (Chart  1).  As  to  the  microscopic  appearance  of  the  culture  it 
was  found  that  no  hard  and  fast  rule  can  be  laid  down  regarding  the  character 
of  the  bacillary  or  coccus  growth.  On  any  medium  the  former  developed  moist 
and  opaque  while  the  latter  grew  more  delicately  and  translucent  or  trans- 
parent. These  characteristics  were  observed  with  the  agar  strain  of  C.  enzy- 
micus  and  are  diametrically  opposed  to  Mellon's  description.  The  strain 
cultured  on  blood  medium  conformed  somewhat  to  his  statement,  yet  transfers 
to  plain  agar  gave  heavy,  moist  growth  as  well  as  the  more  delicate  and  trans- 
lucent. On  fresh  Loeffler's  medium  or  blood  agar  the  coccus  type  could  not  be 
distinguished  macroscopically  from  the  bacillary  form. 

To  settle  the  question  whether  the  coccus  form  is  a  mutant  it  is  necessary 
to  isolate  in  pure  culture  the  two  biotypes  which  cultural  experiments  seem  to 
reveal.  A  true  mutation  must  appear  in  a  pure  line  which  has  been  observed 
over  a  definite  period  and  shown  to  consist  of  a  single  biotype.  As  soon  as 
reversion  occurs  when  environmental  conditions  are  restored  to  normal,  we  are 
no  longer  dealing  with  a  mutation.  Different  types,  to  be  genuine  mutants, 
must  have  developed  when  conditions  remained  constant.  According  to  Mellon 
the  diplococcus  form  of  C.  enzymicus  when  obtained  from  the  barred,  long 
bacillary  type,  remains  quite  constant,  and  although  it  is  possible,  as  he  states, 
to  cause  it  to  assume  a  bacillary  form  again,  this  is  accomplished  with  great 
difficulty.  It  is  obvious  that  these  characteristics  are  not  those  of  a  true  mutant, 
since  the  coccus,  according  to  the  author,  is  not  obtained  until  conditions  are 
changed  and  since  it  may  be  transformed  to  the  bacillary  type  although  this  is 
attended  with  difficulty.  Mellon's  view  is  that  the  organism  is  extremely  pleo- 
morphic and  he  disregards  the  likelihood  of  the  coccus  being  a  mutant  or  a 
distinct  biotype  in  his  original  pure  culture.  It  is  difficult,  admittedly,  to  prove 
that  the  coccus  is  an  entity  in  the  original  culture,  yet  it  can  be  done,  especially 
since  there  are  so  many  factors  in  favor  of  this  conception.  Mellon  goes  on 
to  describe  the  source  of  his  culture  and  says  in  part  "the  rabbit  had  received 
intravenous  injections  of  the  strain  in  bacillary  form  and  was  recovered  from 
the  gallbladder  in  long  chains  of  streptococci."  Is  pleomorphism  the  only  pos- 
sible explanation  for  this  strange  transformation?  It  is  just  as  plausible  and 
simpler  to  account  for  this  by  selective  action  on  the  part  of  the  organs  for 
the  coccus  biotype. 


Diphtheroid  Organisms  39 

Experiments  with  Mellon  Strain  Cultivated  on  Blood  Medium. — This  culture, 
obtained  directly  from  Dr.  Mellon,  was  morphologically  like  a  typical  diph- 
theroid containing  barred,  granular,  split  and  clubbed  forms,  and  was  relatively 
free  from  very  minute  ovoid  bacteria,  so  far  as  could  be  revealed  by  a  routine 
smear  examination.  The  strain  had  been  cultivated  on  blood  agar  for  about 
1  year  and  showed  well-developed  adaptability  to  this  medium. 

Study  with  Pure  Lixes  of  C.  Enzymicus  Bacillary  Type 

Sheep  blood  dextrose  agar  plates  were  smeared  with  a  platinum  loop  lightly 
inoculated  with  the  original  culture.  After  24  hours'  incubation  at  37  C.  single 
colonies  were  taken  for  seeding  a  second  series  of  plates.  This  process  was 
repeated  for  10  generations,  using  in  each  instance  but  1  colony  for  the 
subculture.  A  single  colony,  when  examined  in  a  smear,  always  showed  the 
typical  bacillary  appearance,  yet  was  not  "pure"  insofar  as  type  was  con- 
cerned. From  40  to  50  separate  colonies  selected  from  each  generation  were 
studied  microscopically  and  invariably  ovoid  and  very  small  coccoid  forms 
could  be  found  in  goodly  numbers.  By  carefully  examining  numerous  fields 
the  interesting  fact  is  revealed  that  a  small  diplococcus  or  coccus  type  is 
present.  Painstaking  study  of  very  many  microscopic  fields  is  necessary  before 
passing  judgment  on  the  presence  or  absence  of  these  types,  but  they  can  be 
found  none  the  less.  Whether  or  not  these  diplococci  are  appearances  caused 
by  unusually  short  ovoid  or  coccoid  forms,  I  cannot  say,  since  the  line  of 
demarkation  between  these  types  is  not  marked,  but,  as  seen,  they  were  dis- 
tinctly spherical  and  appeared  in  pairs.  Although  the  contrast  was  sufficiently 
striking  with  a  magnification  of  about  1,500  diameters,  a  high  power  Jens  with 
a  magnification  of  2,500  dispelled  any  doubt.  Where  two  such  organisms  were 
united,  one  could  distinguish  the  point  of  union  from  that  which  is  observed 
in  coccoid  and  small  rod-forms.  With  these  observations  to  go  by,  Mellon's 
contention  regarding  his  bacillary  culture  becomes  untenable,  and  it  is  perhaps 
inaccurate  to  say  that  "a  culture  of  the  C.  enzymicus  was  prepared  from  a 
single  colony  whose  antecedents  had  shown  no  diplococci."  The9retically  as 
well  as  practically  the  culture  is  absolutely  pure  but  we  are  not  justified,  under 
any  circumstances,  in  assuming  that  a  single  biotype  can  be  so  obtained.  It  is 
not  that  it  is  difficult  to  obtain  the  bacillary  form  from  the  coccus,  but 
that  it  is  difficult,  if  not  impossible,  to  get  the  bacillus  without  the  coccus. 
Agai'  slants,  inoculated  with  single  colonies  from  various  generations  plated  out 
according  to  the  technic  described,  always  contained  cocci. 

"Transition"  of  Bacillus  to  Coccus. — The  technic  of  Mellon  was  repeated 
carefully  in  all  details.  Meat  infusion  veal  glucose  broth  1.6  to  phenolphthalein 
was  enriched  with  sterile  rabbit  serum  to  the  amount  of  about  5  c.c.  per  liter. 
Flasks  and  tubes  were  prepared  containing  30  c.c.  and  10  c.c.  of  the  medium, 
respectively,  and  tested  for  sterility  before  use.  A  single  colony  from  the 
8th  generation  of  plates  made  with  the  bacillary  types,  previously  described, 
was  planted  in  a  tube  of  this  medium,  incubated  at  37  C.  for  24  hours  and 
then  at  28  C.  for  3  days.  From  this  tube  the  sediment  was  transferred  to  a 
flask  of  the  same  broth  and  incubated  at  37  C.  After  different  intervals 
smears  were  made  from  supernatant  fluid  and  sediment  and  examined.  At 
no  time  was  any  change  to  a  coccus  observed,  and  the  picture  did  not  differ 
in  any  degree  from  that  seen  under  ordinary  conditions  on  agar  slants. 
Before  we  can  affirm  that  the  morphologic  appearance  represents  the  actual 
mechanism  of  transformation,  it  is  essential  that  a  culture  of  cocci  be  obtained 
and    propagated  for  several  generations   from   material   showing  this   "transi- 


40  Frederick  Eberson 

tional"  picture.  Repeating  Mellon's  technic  I  have  not  succeeded  in  demon- 
strating this  and  I  find  that  his  paper  contains  no  evidence  that  such  a 
postulate  has  been  fulfilled.  Subcultures  were  made  on  agar,  after  12,  24,  30, 
48,  72,  and  96  hours,  from  individual  flasks  which  had  been  seeded  with  the 
sediment  obtained  from  tubes  incubated  at  25-28  C.  for  3  days.  In  none  of 
these  transplants,  incubated  at  Zl  C.  for  24  hours,  was  a  culture  of  cocci 
obtained.  There  was,  however,  an  abundance  of  extremely  short  forms — 
coccoids  and  some  diplococcoids,  hut  nothing  to  indicate  that  the  bacillary  type 
had  undergone  any  definite  change.  When  cocci  are  present,  the  picture  is 
unmistakable  and  there  can  be  no  confusion  of  this  type  with  the  very  short 
forms  which  look  like  diplococci   (Fig.  26).  ' 

It  is  questionable  whether  the  refined  technic  of  Mellon  is  needed  to  demon- 
strate what  is  erroneously  called  a  transformation.  That  all  diphtheroid  cul- 
tures show  two  distinct  forms  of  organisms  is  beyond  question  and  that  these 
types  may  be  seen  under  usual  laboratory  conditions,  is  also  a  matter  of 
common  experience.  Great  caution  is  necessary  in  interpreting  a  morphologic 
appearance  as  the  actual  process  responsible  for  the  occurrence  of  different 
forms  in  bacterial  culture.  Repeated  subcultures  gave  negative  results  and  at 
no  time  was  it  possible  to  show  that  complete  disintegration  had  taken  place. 
The  "mucus-like  debris"  and  "concentration  of  chromatin"  described  by  Mellon 
was  seen  after  24  hours,  not  only  in  broth  cultures  made  according  to  the 
special  technic  described,  but  also  in  agar  slants  grown  under  conditions  to 
be  given.  Transfers  from  broth  containing  "disintegrated  bacillary  forms" 
naturally  failed  to  give  mucus-like  shreds  which  are  found  in  smears,  but 
the  explanation  for  this  is  very  simple.  Detritus  can  hardly  be  expected  to 
grow  in  Tiransplants.  As  for  the  "chromatin  masses  approaching  each  other  in 
transplants  until  the  figure  cannot  be  told  from  a  diplococcus,"  the  fact  remains 
that  these  forms  only  resemble  diplococci.  In  order  to  study  the  effect  of  lower 
temperatures  on  the  possible  suppression  of  bacillary  forms  which  might  occur 
during  the  interval-  when  the  broth  cultures  are  incubated  at  25-30  C,  a  few 
experiments  were  performed  with  agar  cultures. 

Kinyoun's  method  for  the  staining  of  diphtheria  was  used.  This  stain  gives 
most  marked  granular  appearance  and  is  best  adapted  to  differentiating  cocci 
from  bacillary  bodies  with  chromatin  substance  so  disposed  as  to  mislead  one 
into  taking  these  for  true  cocci.  Agar  slants  were  prepared  from  a  bacillary 
culture  of  C.  enzymicus  and  incubated  at  Zl  C.  for  24  hours.  As  usual,  some 
coccus  forms  could  be  discerned  in  smears.  The  picture,  however,  was  bacillary. 
Such  cultures  when  incubated  further  at  room  temperatures,  21-23  C.  for  12 
hours,  showed  in  smears  numerous  diplococci  and  a  marked  overgrowth  of 
very  short  forms.  Subcultures  maintained  at  these  temperatures  always  yielded 
the  same  results.  When  transfers  were  made  to  agar  and  incubated  at  37  C, 
however,  the  diplococci  became  rare  and  finally  could  not  be  found  at  all. 
At  28  C.  the  effect  on  morphology  was  evident  also.  After  48  hours  at  this 
temperature,  diplococci  became  numerous,  and  after  5  days  they  were  present 
in  practically  pure  culture.  Transfers  to  agar  incubated  at  28  and  2il  C.  gave, 
respectively,  numerous  diplococci  and  short  forms  and  few  diplococci.  Broth 
cultures,  kept  at  28  C.  for  1  week,  were  found  to  contain  numerous  diplococci 
and  extremely  short  forms  which  could  not  be  distinguished  readily  from  these. 

Coccus  Type. — A  pure  culture  of  cocci  was  obtained  from  the  original 
diphtheroid  strain,  as  described  previously,  and  attempts  were  made  to  alter 
its  morphology.  The  results  of  the  experiments  showed  conclusively  that 
when  working  with  such  a  pure  strain  of  cocci  it  is  impossible  to  transform 


Diphtheroid  Organisms  41 

it  into  a  bacillus.  In  the  serologic  experiments  described  subsequently  it  is 
shown  that  the  coccus  is  antigenically  distinct  from  the  bacillary  type  and  that 
the  extremely  short  types  which  are  identical  in  appearance  with  cocci  are 
merely  altered  bacillary  forms.  These  "cocci,"  to  be  designated  here  as 
Strain  28,  are  antigenically  the  same  as  the  long,  granular  rods. 

Diphtheroid  cocci  were  grown  on  Loeffler's  serum  for  27  generations,  and  no 
change  was  noted  in  morphology.  The  organisms  remained  cocci.  On  glucose 
serum  medium  the  forms  developed  considerably  larger  and  thicker,  but  were 
typical  diplococci  in  outline.  On  agar  of  various  H-ion  concentrations  ranging 
from  6.1-5.2,  no  change  in  form  was  observed.  The  use  of  potassium  bichro- 
mate in  broth  containing  various  amounts  of  acid  failed  to  alter  the  coccus. 

A  bacillary  strain  of  C.  enzymicus  was  cultivated  on  plain  agar  at  28  C.  for 
several  days  and  subcultures  made  from  day  to  day.  After  one  week  at  this  tem- 
perature, stained  specimens  from  different  slants  showed  forms  which  could 
hardly  be  distinguished  from  diplococci.  These  proved  on  close  examination 
to  be  minute  rods  unevenly  stained  and  bipolars.  Such  cultures,  if  transferred 
to  serum  medium,  assumed  the  typical  bacillary  form.  Figure  18  illustrates 
clearly  how  readily  one  may  err  in  calling  these  forms  cocci.  By  comparing 
them  with  the  true  cocci  shown  in  Figure  16  the  difference  can  be  seen. 

Behavior  of  Coccus  and  Bacillary  Types  in  Carbohydrates. — Sugar  broths 
(1.0+  to  phenolphthalein)  were  made  in  the  usual  manner,  and  duplicate  sets 

TABLE  1 
Acid  Production 

C.  Enzymfcus— Coccus       Control       0.  Enzymicus— Bacillary 

CC  GO  CO 

Dextrose 7.4  1.6  7.0 

Lactose 6.1  1.4  7.1 

Saccharose 7.2  1.7  6.9 

Maltose 8.0  1.8  '         7.8 

Inulin 2.1  1.8  2.0 

Dextrin 3.8  1.4  4.4 

Glycerol 2.4  1.8  7.4 


of  tubes  were  inoculated  from  a  24-hour  old  agar  culture  of  C.  enzymicus 
(coccus)  and  C.  enzymicus  (bacillary).  After  8  days'  incubation  at  37  C.  the 
titrable  acid  formed  in  each  of  the  carbohydrates  was  determined  by  addition 
of  N/20  NaOH,  using  phenolphthalein  as  an  indicator.  Titrations  were  done 
in  the  cold.  Five  c.c.  of  broth  were  used  in  a  total  volume  of  50  c.c.  with 
distilled  HaO. 

Examination  of  the  tubes  after  inoculation  with  both  strains  showed  the 
marked  difference  in  rate  of  growth  of  the  organisms.  After  8  hours  the  coccus 
type  had  clouded  all  of  the  sugar  broths  heavily.  The  bacillary  type  developed 
more  slowly,  and  the  intensity  of  clouding  after  24  hours  approximated  that 
produced  by  the  coccus  in  a  third  of  the  time.  Both  series  showed  fine  granu- 
lation and  sedimentation,  but  this  was  more  pronounced  in  the  case  of  the 
coccus. 

The  acid-production  is  given  in  Table  1.  The  figures  represent  the  amount 
of  N/20  NaOH  required  to  neutralize  acid. 

The  tabulation  shows  at  a  glance  that,  with  the  exception  of  glycerol  and 
dextrin  perhaps,  both  types  behaved  similarly  in  the  carbohydrates  used.  This 
might  be  an  argument  for  similarity  or  relationship  of  both  organisms.  The 
difference  observed  in  glycerol,  however,  was  too  marked  to  escape  notice  and 


42  Frederick  Eberson 

on  the  basis  of  this  alone  would  indicate  a  distinct  property  not  common  to 
both  organisms.  In  order  to  control  the  fermentation  test,  stained  preparations 
were  made  from  both  sets  of  tubes  just  before  titrating,  and  the  apparent 
similarity  of  reaction  was  explained  by  the  selective  action  of  certain  mediums 
on  the  type  of  organism.  Glycerol  was  found  to  inhibit  the  development  of 
cocci  completely;  dextrin  had  a  similar  action  to  a  lesser  degree.  In  all  of 
the  remaining  carbohydrates,  the  bacillary  forms  had  been  suppressed  to  a 
remarkable  extent. 

Serologic    Experiments 

Absorption  of  Agglutinins. — The  object  of  these  experiments  was  to  estab- 
lish, if  possible,  by  means  of  highly  agglutinating  serums,  the  relationship 
of  the  bacillary  diphtheroid  to  the  diplococcus.  Mellon,  in  his  paper,  concludes 
that  the  streptococci  are  related  to  the  diphtheroids  and  can  be  derived  from 
these.  To  establish  such  a  claim  it  would  be  necessary  to  obtain  pure  strains 
of  each  of  these  two  types  and  show  that  they  are  mutually  interchangeable. 
This  has  not  been  shown  by  Mellon,  and  in  the  work  reported  here  the  results 

TABLE  2 
Absorption    Experiments   with    Coccus    Serum 

Serum  C.  Enzymicus— Coccus 

1-20    1-40    1-80    1-160    1-320    1-500  1-1000    1-1500    1-2000    1-3000    1-4000 

C.  enzymicus  (coccus)                               ±       +       ++     ++  ++       ++       +-|-        4.         -+- 
C.  enzymicus  (bacillary)          —      —      —       —        —        — 

After  absorption  with  C.  enzymicus  (bacillary) 

C.  enzymicus  (coccus)                                                  +.f4--t-  ++       ++       ++        +          -j- 
C.  enzymicus  (bacillary)          —      —      —       —         —        -^ 

After  absorption  with  C.  enzymicus— Strain  28 

0.  enzymicus  (coccus)                                                  -^-^^--f  ++      ++       ++        +         ± 

C.  enzymicus  (28  strain)          ______  _ 


do  not  substantiate  his  claim.  It  is  evident  that  two  organisms,  derived  one 
from  the  other,  should  have  similar,  if  not  identical,  antigenic  properties.  To 
test  the  validity  of  such  an  assumption  the  most  delicate  method  available  is 
that  of  agglutinin  absorption,  when  the  organisms  in  question  are  capable  of 
producing  agglutinating  serums.  If  the  coccus  type,  obtained  from  a  diph- 
theroid culture,  is  truly  derived  from  the  bacillus,  then  a  serum  prepared  with 
one  strain  should  agglutinate  the  other,  and  the  absorption  of  agglutinins 
should  be  demonstrable.  With  this  idea  in  view,  two  serums  were  prepared 
with  the  coccus  strain  and  the  bacillary  strain,  respectively. 

Healthy  male  rabbits  were  injected  intravenously  at  4-6  day  intervals 
with  increasing  doses  of  culture  heated  to  56  C.  for  20  minutes.  The  dosage 
progressed  from  l^o  of  an  agar  slant  to  a  whole  culture.  Ten  days  after  the 
last  injection  the  animals  were  exsanguinated  and  the  serum,  after  heating  to 
53  C.  for  15  minutes,  was  stored  at  4  C.  until  ready  for  use.  The  tests  were 
made  with  serum  less  than  6  days  old. 

The  technic  of  absorption  was  as  follows :  24-hour  old  agar  cultures  of 
the  organisms  were  emulsified  in  a  small  volume  of  NaCl  solution,  heated 
to  56  C.  for  20  minutes,  and  centrifugated  at  high  speed  for  a  short  time. 
To  the  sediment  so  obtained  the  heterologous  serum  was  added  after  deter- 
mining by  a  preliminary  agglutination  test  the  potency  of  the  homologous 
serum.     In  order  to  ensure  complete  absorption  the  serums  were  diluted.     The 


Diphtheroid  Organisms  43 

mixture  of  bacteria  and  serum  was  incubated  at  TH  C.  for  IVa  hours,  kept  at 
4  C.  over  night,  and  then  centrifugated  for  20  minutes  at  high  speed.  The 
supernatant  serum,  pipetted  off,  was  added  to  another  heavy  sediment  of 
organisms,  obtained  as  in  the  preceding,  and  the  absorption  repeated,  after 
which  agglutination  tests  were  made.  A  series  of  dilutions  were  prepared 
with  each  original  serum  and  tubes  set  up  at  the  same  time  as  the  absorbed 
serum. 

The  results  of  these  absorption  experiments  are  decisive.  A  serum 
prepared  with  the  coccus  possesses  no  agglutinins  for  the  bacillary 
diphtheroid  and  when  absorbed  completely  with  this  bacillary  strain 
evidences  no  reduction  in  its  agglutination  titer  against  the  homologous 
coccus.  The  coccus  serum  when  absorbed  with  a  bacillary  strain  altered 
morphologically  by  cultivation  at  28  C.  for  10  days  so  that  the  organism 

TABLE  3 
Absorption    Experiments    with    Bacillary    Serum 

Serum  C.  Enzymicus— Bacillary 

1-10    1-20    1-50     1-100      1-20O      1-400      1-800     1-1600    1-20CO    1-30OO 
0.  enzymicus   (bacillary)  ++        ++        ++         +  ±  — 

C.  enzymicus  (coccus)  +       —       —        —  —  — 

After   absorption  with   C.  enzymicus   (coccus) 
C.  enzymicus   (bacillary)                            ++++        ++        ++         +,  ± 

After  absorption  with  C.  enzymicus— Strain  28 
C.  enzymicus   (bacillary)  ±         ++         +  —  — 


could  not  be  distinguished  from  cocci,  lost  none  of  its  agglutinating 
power  for  the  coccus.  Again  evidence  is  adduced  that  the  change  on 
the  part  of  the  bacillus  was  merely  an  appearance,  and  that  the  organ- 
isms were  not  true  cocci,  but  extremely  short  bacilli,  altered  perhaps  by 
changes  in  surface  tension  to  assume  a  form  remarkably  like  cocci. 
Antigenically  these  organisms  are  therefore  distinct  from  the  coccus 
type.  In  Table  3  they  are  shown  to  be  identical  with  the  original  bacil- 
lary form.  The  conclusion  to  be  drawn  from  this  experiment  is  that 
the  coccus  has  no  antigenic  elements  in  common  with  the  bacillus. 

The  next  experiment,  made  with  a  serum  produced  with  the  bacil- 
lary strain  of  C.  enzymicus,  gave  additional  evidence  in  another  direc- 
tion and  confirmed  the  difference  of  the  two  organisms.  The  serum, 
although  agglutinating  its  homologous  culture  as  high  as  1-2000,  gave 
agglutination  in  1-10  for  the  coccus.  This  in  itself  would  be  an  argu- 
ment against  antigenic  relationship.  The  fact  that  a  bacillary  strain  is 
never  free  from  cocci,  however,  would  readily  explain  slight  agglutina- 
tion which  might  occur.     This  interesting  point  is  shown  quite  clearly 


44  Frederick  Eberson 

in  the  absorption  test.  The  serum,  after  complete  absorption  with  the 
coccus,  showed  a  reduction  in  agglutinating  titer,  stopping  at  1-1600, 
whereas  the  nonabsorbed  serum  was  positive  at  1-2000.  This  removai 
of  agglutinins  might  suggest  antigenic  similarity  on  the  part  of  the 
coccus  for  the  bacillus.  The  important  point  to  be  noted  here,  how- 
ever, is  the  cumulative  action  of  multiple  doses  of  bacillary  diphtheroids 
containing  cocci  in  mixture.  Repeated  injections  of  mass  cultures 
have  resulted  in  the  formation  of  agglutinins  specific  for  each  type  of 
organism,  and  the  slight  reduction  in  titer  is  thereby  accounted  for.  As 
a  further  corollary,  Strain  28  absorbs  most  of  the  agglutinins  out  of 
the  bacillary  serum,  showing  that  the  organisms  are  antigenically  the 
same.  That  the  absorption  is  not  more  complete  may  be  due  to  the 
fact  that  but  one  exposure  was  made  with  the  organism  in  question, 
whereas  in  the  case  of  the  coccus  strain  the  serum  was  doubly  absorbed. 
From  these  findings  the  conclusion  may  be  drawn  that  the  bacillus  is 
antigenically  distinct  from  the  coccus. 

Two  possibilties  are  suggested  in  explanation  of  the  presence  side 
by  side  of  two  biotypes ;  one  is  that  of  mutation  and  the  second  a 
symbiotic  relationship  of  a  contaminating  coccus  and  a  typical  bacillary 
diphtheroid.  The  possibility  of  mutation  is  not  far-fetched,  although 
highly  improbable  in  this  instance.  As  to  a  symbiosis,  the  likelihood 
is  less  remote.  In  either  case  the  question  can  be  answered  categorically 
by  applying  the  single  bacterial-cell  method  to  the  conditions  which 
prevailed  in  the  experiments  given  here.  These  experiments  will  be 
described  in  a  future  paper. 

SUMMARY     AND     CONCLUSIONS 

The  diphtheroids  have  been  studied  with  regard  to  nomenclature 
and  revised  according  to  accepted  standards  for  the  naming  of  valid 
species. 

A  tentative  classification  is  offered  with  a  view  toward  grouping 
prominent  biologic  characters.  Nine  distinct  groups  of  diphtheroids 
are  outlined  with  a  type  species  for  each  group  and  corresponding 
subsf>ecies. 

It  is  evident  that  the  groups  hitherto  accepted  as  types  for  all  diph- 
theroids are  insufficient  and  not  representative  of  a  broad  classification. 

Diphtheroids  isolated  from  glands  and  tissues — 21  different  sources 
— fall  into  12  distinct  fermentative  groups.  The  greatest  number  of 
organisms  are  nonfermenters.     Strains  isolated  from  the  eye  and  nose 


Diphtheroid  Organisms  45 

do  not  attack  carbohydrates  vigorously,  and  are  usually  found  either  in 
the  nonfermenting  or  in  the  dextrose-splitting  group. 

The  diphtheroids  associated  with  Hodgkin's  disease  have  been 
studied.  It  has  been  shown  that  neither  the  source  nor  the  cultural 
characters  serve  to  distinguish  the  supposed  cause  of  Hodgkin's  disease 
from  numerous  saprophytic  diphtheroids.  By  means  of  complement 
fixation  tests  the  conclusion  is  reached  that  the  cause  of  this  disease  is 
not  the  organism  described  by  numerous  workers. 

Agglutinin  absorption  studies  indicate  more  clearly  the  relationship 
which  exists  between  certain  members  of  the  diphtheroid  group  of  bac- 
teria.    The  results  are  correlated  in  the  main  with  complement  fixation. 

Diphtheroids  conform  to  the  fundamental  laws  of  bacteriology  and 
are  not  readily  transformed  into  cocci  and  back  again.  Two  distinct 
biotypes  have  been  isolated  from  a  culture  of  C.  enzymicus. 

The  coccus  associated  with  a  bacillary  diphtheroid  (C.  enzymicus) 
is  antigenically  distinct  from  the  bacillus  and  vice  versa.  Under  cer- 
tain conditions  a  bacillary  strain  may  be  made  to  assume  a  diplococcus 
picture,  but  absorption  experiments  show  that  the  organisms  are  not 
cocci. 

Certain  mediums  and  cultural  technic  have  been  found  to  exert  a 
definite  selection  on  diphtheroid  cultures.  One  or  the  other  biotype 
will  prevail,  depending  on  the  extent  of  suppression  of  the  first  or 
second. 

Two  explanations  for  the  existence  of  coccus  and  bacillus  are  sug- 
gested, either  that  of  mutation  or  a  symbiotic  relationship  of  a  contam- 
inating coccus.  The  question  can  be  settled  by  applying  the  single-cell 
method  to  the  study. 

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46  Frederick  Ederson 

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Diphtheroid  Organisms 


47 


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48  Frederick  Eberson 


EXPLANATION   OF   PLATES    1-3 
Fig-  1. — C.  acidum.     Large   forms  developing  in  mediums  enriched  with  serum.      X    1000. 
Fig.  2. — Same  after  prolonged  cultivation   on  ordinary  mediums.      X    1000. 
Fig.  3. — C.  suppuratum.     On  serum  medium  after  1st  isolation.      X    1000. 
F'g-  4. — Same  after  prolonged  cultivation  on  ordinary  medium.      X   1000. 
Fig.  5. — C.  aurantiacum.      X    1000. 
Fig.  6. — C.    delicatum.      X    1000. 
Fig.  7. — C.   epidermidis.      X    1000. 
Fig.  8. — C.  ascites.     X   1000. 
Fig.  9. — C.  glandulae.      X    1000. 
Fig.   10. — C.  pseudodiphtheriae.      X    1000. 
Fig.   11. — C.  cerebralis.      X    1000. 
Fig.   12.— C.  putidum.     X    1000. 
Fig.   13. — C.  flocculens.      X    1000. 

Fig.  14.— I.  Plain  agar  slant  inoculated  with  material  taken  from  a  culture  of  C.  enzymicus 
grown  on  sheep  blood  glucose  agar  for  4  generations.  The  sheep  blood  agar  showed  numerous 
rod  forms  with  cocci  intermingled.  A.^  heavy  white  growth  superimposed  on  a  delicate  trans- 
parent background. 

II.  Plain  agar  slant  inoculated  with  material  taken  from  opaque,  heavy  white  growth 
indicated  by^'.  A,  transparent  surface  growth.  B,  heavy  white  opaque  growth.  Smears 
from  A^  and  B  always  showed  bacillary  forms,  typical  diphtheroids.  A  yielded  pure  cocci 
only.     Natural  size. 

Fig.  15. — Smear  from  heavy  growth  indicated  by  /4'  and  B  in  Figure  14. 

Fig.  16. — Smear  from  transparent  delicate  growth  marked  A  in  Figure  14.  Stained  with 
Loeffler's  methylene  blue.     X    1000. 

Fig.  17. — C.  enzymicus.     Original  culture. 

Fig.l8.— Bacillary  type  developing  on  Loeffler's  serum.  Transferred  from  sheep  blood 
agar  on  which  the  original  culture  had  been  inoculated.  When  the  original  strain  (Fig.  17) 
was  planted  on  plain  agar,  bacilli  could  not  be  demonstrated. 

Fig.  19. — Suppression  of  bacillary  type  on  glucose  agar.  After  3  generations  on  Loeffler's 
serum  preceded  by  2  generations  on  sheep  blood  agar,  cocci  were  rare.  Note  the  predom- 
inating type  here. 

Fig.  20. — Transplant  from  3rd  generation  on  sheep  blood  medium  to  plain  agar.  The 
sheep  blood  culture  showed  rare  cocci  and  practically  a  pure  culture  of  bacillary  forms. 

Fig.  21. — Same  culture  before  transplanting  to  plain  agar.      X    1000. 

Fig.  22. — C.  enzymicus  cultivated  on  blood  agar,  then  transferred  to  plain  agar  and  incu- 
bated at  28  C.      "Concentration  of  chromatin"  clearly  shown. 

Fig.  23. — Transplant  from  48-hour  veal  glucose  broth  culture  of  C.  enzymicus.  Note 
absence  of  "transformed"  bacilli.     The  technic  according  to  Mellon. 

Fig.  24. — C.  enzymicus  grown  at  28  C.  in  broth.     Curious  chromatin  staining. 

Fig.  25. — C.  enzymicus  3rd  generation  on  agar  from  blood  medium.  This  picture,  as  well 
as  Figure  22,  is  identical  with  the  "transitional"  phase. 

Fig.  26. — C.  enzymicus  grown  at  28  C.  on  plain  agar  for  10  days.  Note  the  diplococcus 
appearance. 

Fig.  27. — Previous  incubation  at  28  C.  in  broth  and  subsequent  growth  at  37  C.  Stained 
according  to   Kinyoun's  method.      X    1000. 


Plate  1 


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)^,^X^?^ 


t(l*3p^v^'     •■'V- 


<.xi.&^--- 


4 


'"•^y  »  ' 


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^ 
W 


1v  A  . 


-»      tfs".^* 


2 


'^^  >*•% 


^y  474^ 


•c*^ 


^ 


^4'     • 


^A 


y. 


<*»      it 


V".-': 


^ 


I 


r^i 


I 


^ 


12 


•»    f 


13 


Plate  2 


'/^■, 


:«y 


Plate  3 


VITA 

Frederick  Eberson  was  born  in  New  York  City,  February  10, 
1892.  He  received  his  elementary  and  high  school  education  in  the 
city  of  Brooklyn,  N.  Y.,  and  his  first  college  training  at  the  College 
of  the  City  of  New  York,  where  he  took  the  degree  of  Bachelor  of 
Science  in  June,  1912.  In  October,  1912,  he  was  appointed  Research 
j\ssistant  in  the  Department  of  Sanitary  Science  and  Public  Health 
at  the  Massachusetts  Institute  of  Technology,  Boston.  Thence  he 
matriculated  under  the  Faculty  of  Medicine  and  Pure  Science  at 
Columbia  University,  specializing  in  Bacteriology  and  Biological 
Chemistry,  and  received  his  Master  degree  in  June,  1914.  In  Septem- 
ber, 1914,  he  was  appointed  Research  Fellow  and  Assistant  Instructor 
in  the  Department  of  Bacteriology  and  Hygiene  at  the  Iowa  State 
College,  where  he  specialized  in  Bacteriology  and  Pathology  and 
obtained  his  Master  of  Science  degree  in  June,  1915.  In  September 
of  the  same  year  he  was  appointed  Research  Scholar  under  the  tenure 
of  a  President's  University  Scholarship  at  Columbia  University  and 
continued  work  in  the  Department  of  Bacteriology  until  1916,  when 
he  was  appointed  Research  Bacteriologist  to  the  North  Manchurian 
Plague  Prevention  Service,  Harbin,  China,  and  was  placed  in  charge 
of  the  Division  of  Bacteriology  and  Pathology.  Toward  the  end  of 
1917  he  was  appointed  to  the  stafif  of  the  Rockefeller  Institute  for 
Medical  Research  in  the  Department  of  Bacteriology  and  Pathology. 

Publications 

1912 — The   Effect  of   Drying  on  the  Viability  of   Bacteria    (with    C.   E.   A. 

Winslow)  Proceedings  Soc.  for  Exp.  Biol,  and  Medicine,  May  5. 
1915 — A  Bacteriologic  Study  of  Secondary  Invaders  in  Hog  Cholera.    Jour- 
nal of  Infectious  Diseases,  Vol.  17,  No.  2. 
Separation  of  the  Antibody  Fractions  in  Hog  Cholera   Serum.     Jour- 
nal of  Infectious  Diseases,  Vol.  17,  No.  2. 
A  Milk-Borne  Paratyphoid  Outbreak  in  Ames,  Iowa  (with  M.  Levine). 
Journal  of  Infectious  Diseases,  Vol.  18,  No.  2. 
1917 — Plague  Poisons  and  Virulence.     Journal  of  Infectious  Diseases,  Vol. 
20,  No.  2;  National  Medical  Journal  of  China,  Vol.  3,  No.  1. 
Transmission  of   Pneumonic  and   Septicemic   Plague  among   Marmots. 
Journal  of  Infectious  Diseases,  Vol.  20,  No.  2;  National   Medical 
Journal    of    China,    Vol.    3,    No.    2;    English    Journal    of    Hygiene. 
Vol.  16.  No.  1. 


56  Frederick  Iu^erson 

Nature    of     Plague     Proteotoxius.      Journal    of    Infectious    Diseases, 
Vol.  21,  No.  1 ;  National  Medical  Journal  of  China.  Vol.  3.  No.  l' 
1918 — Active  Immunity  to  Systemic  Plague  Infection.    Journal  of  Infectious 
Diseases,  Vol.  22,  p.  62. 

Experiments  with  Rosenow's  Antipoliomyelitic  Serum  (with  H.  L. 
Amoss).  Journal  of  Experimental  Medicine.  Vol.  27,  No.  2;  Proc 
Soc.  for  Exp.  Biol,  and  Medicine.  April. 

Physiological  Study  of  the  Choroid  Plexus  and  Experimental  I 'olio- 
myelitis  (with  Simon  Flexner  and  H.  L.  Amoss),  Journal  of 
Experimental  Medicine,  Vol.  27,  No.  6. 

Experiments  with  Nuzum  and  Willy's  Antipoliomyelitic  Serum  (with 
H.  L.  Amoss).    Journal  of  Experimental  Medicine,  Vol.  28,  No.  1.; 

Effect  of  Carbohydrate  Concentration  on  Acid  Production.  Biochemical 
Bulletin   (in  press). 

Scientific  Societies 
Society  of  American  Bacteriologists. 

American   Association   for   the  Advancement   of   Science. 
National  Medical  Association  of  China. 
Biochemical  Society,  Columlna  University. 


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